Muteins of tear lipocalin and methods for obtaining the same

ABSTRACT

The present invention relates to novel muteins derived from human tear lipocalin. The invention also refers to a corresponding nucleic acid molecule encoding such a mutein and to a method for its generation. The invention further refers to a method for producing such a mutein. Finally, the invention is directed to a pharmaceutical composition comprising such a lipocalin mutein as well as to various uses of the mutein.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/616,815, filed Sep. 14, 2012, which is a Divisional of U.S.application Ser. No. 12/309,820 filed Jun. 11, 2009 which is the USNational Stage of PCT/EP2007/057971 filed Aug. 1, 2007 which claims thebenefit of priority from U.S. Provisional Application 60/821,073, filedAug. 1, 2006 and U.S. Provisional Application 60/912,013, filed Apr. 16,2007. All of the aforesaid applications are incorporated herein byreference in their entirety.

STATEMENT CONCERNING THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 2, 2016, isnamed sequence.txt and is 103 KB.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel muteins derived from human tearlipocalin that bind a given non-natural ligand with detectable affinity.The invention also relates to corresponding nucleic acid moleculesencoding such a mutein and to a method for their generation. Theinvention further relates a method for producing such a mutein. Finally,the invention is directed to a pharmaceutical composition comprisingsuch a lipocalin mutein as well as to various uses of the mutein.

2. Description of Related Art

The members of the lipocalin protein family (Pervaiz, S., and Brew, K.(1987) FASEB J. 1, 209-214) are typically small, secreted proteins whichare characterized by a range of different molecular-recognitionproperties: their ability to bind various, principally hydrophobicmolecules (such as retinoids, fatty acids, cholesterols, prostaglandins,biliverdins, pheromones, tastants, and odorants), their binding tospecific cell-surface receptors and their formation of macromolecularcomplexes. Although they have, in the past, been classified primarily astransport proteins, it is now clear that the lipocalins fulfill avariety of physiological functions. These include roles in retinoltransport, olfaction, pheromone signaling, and the synthesis ofprostaglandins. The lipocalins have also been implicated in theregulation of the immune response and the mediation of cell homoeostasis(reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).

The lipocalins share unusually low levels of overall sequenceconservation, often with sequence identities of less than 20%. In strongcontrast, their overall folding pattern is highly conserved. The centralpart of the lipocalin structure consists of a single eight-strandedanti-parallel β-sheet closed back on itself to form a continuouslyhydrogen-bonded β-barrel. One end of the barrel is sterically blocked bythe N-terminal peptide segment that runs across its bottom as well asthree peptide loops connecting the β-strands. The other end of theβ-barrel is open to the solvent and encompasses a target-binding site,which is formed by four peptide loops. It is this diversity of the loopsin the otherwise rigid lipocalin scaffold that gives rise to a varietyof different binding modes each capable of accommodating targets ofdifferent size, shape, and chemical character (reviewed, e.g., inFlower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, orSkerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).

Human tear pre-albumin, now called tear lipocalin (TLPC or Tlc), wasoriginally described as a major protein of human tear fluid(approximately one third of the total protein content) but has recentlyalso been identified in several other secretory tissues includingprostate, nasal mucosa and tracheal mucosa. Homologous proteins havebeen found in rat, pig, dog and horse. Tear lipocalin is an unusuallipocalin member because of its high promiscuity for relative insolublelipids and binding characteristics that differ from other members ofthis protein family (reviewed in Redl, B. (2000) Biochim. Biophys. Acta1482, 241-248). A remarkable number of lipophilic compounds of differentchemical classes such as fatty acids, fatty alcohols, phospholipids,glycolipids and cholesterol are endogenous ligands of this protein.Interestingly, in contrast to other lipocalins the strength of ligand(target) binding correlates with the length of the hydrocarbon tail bothfor alkyl amides and fatty acids. Thus, tear lipocalin binds moststrongly the least soluble lipids (Glasgow, B. J. et al. (1995) Curr.Eye Res. 14, 363-372; Gasymov, O. K. et al. (1999) Biochim. Biophys.Acta 1433, 307-320).

The precise biological function of human tear lipocalin has not beenfully elucidated so far and is still a matter of controversy. In tearfluid, it appears to be most important for the integrity of the tearfilm by removing lipids from the mucous surface of the eye to the liquidphase (reviewed in Gasymov, O. K. et al. (1999), supra). However, itdisplays additional activities in vitro that are very unusual amonglipocalins, namely inhibition of cysteine proteinases as well asnon-specific endonuclease activity (van't Hof, W. et al. (1997) J. Biol.Chem. 272, 1837-1841; Yusifov, T. N. et al. (2000) Biochem. J. 347,815-819). Recently, it has been demonstrated that tear lipocalin is ableto bind several lipid peroxidation products in vitro resulting in thehypothesis that it might function as a physiologicaloxidative-stress-induced scavenger of potentially harmful lipophilicmolecules (Lechner, M. et al. (2001) Biochem. J. 356, 129-135).

Proteins, which selectively bind to their corresponding targets by wayof non-covalent interaction, play a crucial role as reagents inbiotechnology, medicine, bioanalytics as well as in the biological andlife sciences in general. Antibodies, i.e. immunoglobulins, are aprominent example of this class of proteins. Despite the manifold needsfor such proteins in conjunction with recognition, binding and/orseparation of ligands/targets, almost exclusively immunoglobulins arecurrently used. The application of other proteins with definedligand-binding characteristics, for example the lectins, has remainedrestricted to special cases.

Rather recently, members of the lipocalin family have become subject ofresearch concerning proteins having defined ligand-binding properties.The PCT publication WO 99/16873 discloses polypeptides of the lipocalinfamily with mutated amino acid positions in the region of the fourpeptide loops, which are arranged at the end of the cylindrical β-barrelstructure encompassing the binding pocket, and which correspond to thosesegments in the linear polypeptide sequence comprising the amino acidpositions 28 to 45, 58 to 69, 86 to 99, and 114 to 129 of thebilin-binding protein of Pieris brassicae.

The PCT publication WO 00/75308 discloses muteins of the bilin-bindingprotein, which specifically bind digoxigenin, whereas the InternationalPatent Applications WO 03/029463 and WO 03/029471 relate to muteins ofthe human neutrophil gelatinase-associated lipocalin (hNGAL) andapolipoprotein D, respectively. In order to further improve and finetune ligand affinity, specificity as well as folding stability of alipocalin variant various approaches using different members of thelipocalin family have been proposed (Skerra, A. (2001) Rev. Mol.Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys.Chem. 96, 213-228), such as the replacement of additional amino acidresidues. The PCT publication WO 2006/56464 discloses muteins of humanneutrophile gelatinase-associated lipocalin with binding affinity forCTLA-4 in the low nanomolar range.

The PCT publication WO 2005/19256 discloses muteins of tear lipocalinwith at least one binding site for different or the same target ligandand provides a method for the generation of such muteins of human tearlipocalin. According to this PCT application, certain amino acidstretches within the primary sequence of tear lipocalin, in particularthe loop regions comprising amino acids 7-14, 24-36, 41-49, 53-66,69-77, 79-84, 87-98, and 103-110 of mature human tear lipocalin, aresubjected to mutagenesis in order to generate muteins with bindingaffinities. The resulting muteins have binding affinities for theselected ligand (K_(D)) in the nanomolar range, in most cases>100 nM.

BRIEF SUMMARY OF THE INVENTION

Despite this progress it would be still desirable to have a method forthe generation of human tear lipocalin muteins that possess improvedbinding properties for a selected target molecule, for example in thepicomolar range, simply for the reason to further improve thesuitability of muteins of human tear lipocalin in diagnostic andtherapeutic applications.

Accordingly, it is an object of the invention to provide human tearlipocalin muteins having high binding affinity for a given target.

This object is accomplished by a method for the generation of a humantear lipocalin mutein having the features of the independent claims.

In a first aspect, the present invention provides a method for thegeneration of a mutein of human tear lipocalin, wherein the mutein bindsa given non-natural ligand of human tear lipocalin with detectablebinding affinity, including:

-   -   (a) subjecting a nucleic acid molecule encoding a human tear        lipocalin to mutagenesis at at least one codon of any of the        amino acid sequence positions 26-34, 56-58, 80, 83, 104-106 and        108 of the linear polypeptide sequence of native mature human        tear lipocalin, wherein at least one of the codons encoding        cysteine residues at sequence positions 61 and 153 of the linear        polypeptide sequence of the mature human tear lipocalin has been        mutated to encode any other amino acid residue, thereby        obtaining a plurality of nucleic acids encoding muteins of human        tear lipocalin,    -   (b) expressing the one or more mutein nucleic acid molecule(s)        obtained in (a) in an expression system, thereby obtaining one        or more mutein(s), and    -   (c) enriching the one or more mutein(s) obtained in step (b) and        having detectable binding affinity for a given non-natural        ligand of human tear lipocalin by means of selection and/or        isolation.

In this context it is noted that the inventors have surprisingly foundthat removal of the structural disulfide bond (on the level of arespective nave nucleic acid library) of wild type tear lipocalin thatis formed by the cystein residues 61 and 153 (cf. Breustedt, et al.(2005), The 1.8-Å crystal structure of human tear lipocalin reveals anextended branched cavity with capacity for multiple ligands. J. Biol.Chem. 280, 484-493) provides tear lipocalin muteins that are not onlystably folded but in addition are also able to bind a given non-naturalligand with affinity in the low picomolar range. Without wishing to bebound by theory, it is also believed that the elimination of thestructural disulde bond provides the further advantage of allowing forthe (spontaneous) generation or deliberate introduction of non-naturalartificial disulfide bonds into muteins of the invention (see Examples),thereby increasing the stability of the muteins, for example.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of human tear lipocalin (Swiss-Prot data bankentry P31025) can be substituted by at least one amino acid that is notpresent at this specific position in the respective natural polypeptidesequence. The term “mutagenesis” also includes the (additional)modification of the length of sequence segments by deletion or insertionof one or more amino acids. Thus, it is within the scope of theinvention that, for example, one amino acid at a chosen sequenceposition is replaced by a stretch of three random mutations, leading toan insertion of two amino acid residues compared to the length of therespective segment of the wild type protein. Such an insertion ofdeletion may be introduced independently from each other in any of thepeptide segments that can be subjected to mutagenesis in the invention.In one exemplary embodiment of the invention, an insertion of severalmutations may be introduced into the loop AB of the chosen lipocalinscaffold (cf. International Patent Application WO 2005/019256 which isincorporated by reference its entirety herein). The term “randommutagenesis” means that no predetermined single amino acid (mutation) ispresent at a certain sequence position but that at least two amino acidscan be incorporated with a certain probability at a predefined sequenceposition during mutagenesis.

The coding sequence of human tear lipocalin (Redl, B. et al. (1992) J.Biol. Chem. 267, 20282-20287) is used as a starting point for themutagenesis of the peptide segments selected in the present invention.For the mutagenesis of the recited amino acid positions, the personskilled in the art has at his disposal the various established standardmethods for site-directed mutagenesis (Sambrook, J. et al. (1989),supra). A commonly used technique is the introduction of mutations bymeans of PCR (polymerase chain reaction) using mixtures of syntheticoligonucleotides, which bear a degenerate base composition at thedesired sequence positions. For example, use of the codon NNK or NNS(wherein N=adenine, guanine or cytosine or thymine; K=guanine orthymine; S=adenine or cytosine) allows incorporation of all 20 aminoacids plus the amber stop codon during mutagenesis, whereas the codonVVS limits the number of possibly incorporated amino acids to 12, sinceit excludes the amino acids Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val frombeing incorporated into the selected position of the polypeptidesequence; use of the codon NMS (wherein M=adenine or cytosine), forexample, restricts the number of possible amino acids to 11 at aselected sequence position since it excludes the amino acids Arg, Cys,Gly, Ile, Leu, Met, Phe, Trp, Val from being incorporated at a selectedsequence position. In this respect it is noted that codons for otheramino acids (than the regular 20 naturally occurring amino acids) suchas selenocystein or pyrrolysine can also be incorporated into a nucleicacid of a mutein. It is also possible, as described by Wang, L., et al.(2001) Science 292, 498-500, or Wang, L., and Schultz, P. G. (2002)Chem. Comm. 1, 1-11, to use “artificial” codons such as UAG which areusually recognized as stop codons in order to insert other unusual aminoacids, for example o-methyl-L-tyrosine or p-aminophenylalanine.

The use of nucleotide building blocks with reduced base pairspecificity, as for example inosine, 8-oxo-2′ deoxyguanosine or6(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimindo-1,2-oxazine-7-one(Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another optionfor the introduction of mutations into a chosen sequence segment.

A further possibility is the so-called triplet-mutagenesis. This methoduses mixtures of different nucleotide triplets, each of which codes forone amino acid, for incorporation into the coding sequence (Virnekas B,Ge L, Plückthun A, Schneider K C, Wellnhofer G, Moroney S E. 1994Trinucleotide phosphoramidites: ideal reagents for the synthesis ofmixed oligonucleotides for random mutagenesis. Nucleic Acids Res 22,5600-5607).

One possible strategy for introducing mutations in the selected regionsof the respective polypeptides is based on the use of fouroligonucleotides, each of which is partially derived from one of thecorresponding sequence segments to be mutated. When synthesizing theseoligonucleotides, a person skilled in the art can employ mixtures ofnucleic acid building blocks for the synthesis of those nucleotidetriplets which correspond to the amino acid positions to be mutated sothat codons encoding all natural amino acids randomly arise, which atlast results in the generation of a lipocalin peptide library. Forexample, the first oligonucleotide corresponds in its sequence—apartfrom the mutated positions—to the coding strand for the peptide segmentto be mutated at the most N-terminal position of the lipocalinpolypeptide. Accordingly, the second oligonucleotide corresponds to thenon-coding strand for the second sequence segment following in thepolypeptide sequence. The third oligonucleotide corresponds in turn tothe coding strand for the corresponding third sequence segment. Finally,the fourth oligonucleotide corresponds to the non-coding strand for thefourth sequence segment. A polymerase chain reaction can be performedwith the respective first and second oligonucleotide and separately, ifnecessary, with the respective third and fourth oligonucleotide.

The amplification products of both of these reactions can be combined byvarious known methods into a single nucleic acid comprising the sequencefrom the first to the fourth sequence segments, in which mutations havebeen introduced at the selected positions. To this end, both of theproducts can for example be subjected to a new polymerase chain reactionusing flanking oligonucleotides as well as one or more mediator nucleicacid molecules, which contribute the sequence between the second and thethird sequence segment. In the choice of the number and arrangementwithin the sequence of the oligonucleotides used for the mutagenesis,the person skilled in the art has numerous alternatives at his disposal.

The nucleic acid molecules defined above can be connected by ligationwith the missing 5′- and 3′-sequences of a nucleic acid encoding alipocalin polypeptide and/or the vector, and can be cloned in a knownhost organism. A multitude of established procedures are available forligation and cloning (Sambrook, J. et al. (1989), supra). For example,recognition sequences for restriction endonucleases also present in thesequence of the cloning vector can be engineered into the sequence ofthe synthetic oligonucleotides. Thus, after amplification of therespective PCR product and enzymatic cleavage the resulting fragment canbe easily cloned using the corresponding recognition sequences.

Longer sequence segments within the gene coding for the protein selectedfor mutagenesis can also be subjected to random mutagenesis via knownmethods, for example by use of the polymerase chain reaction underconditions of increased error rate, by chemical mutagenesis or by usingbacterial mutator strains. Such methods can also be used for furtheroptimization of the target affinity or specificity of a lipocalinmutein. Mutations possibly occurring outside the segments ofexperimental mutagenesis are often tolerated or can even prove to beadvantageous, for example if they contribute to an improved foldingefficiency or folding stability of the lipocalin mutein.

The term “human tear lipocalin” as used herein to refer to the maturehuman tear lipocalin with the SWISS-PROT Data Bank Accession NumberP31025.

The term “non-natural ligand” refers to a compound, which does not bindto native mature human tear lipocalin under physiological conditions.The target (ligand) may be any chemical compound in free or conjugatedform which exhibits features of an immunological hapten, a hormone suchas steroid hormones or any biopolymer or fragment thereof, for example,a protein or protein domain, a peptide, an oligodeoxynucleotide, anucleic acid, an oligo- or polysaccharide or conjugates thereof, a lipidor another macromolecule.

In one embodiment of the invention, the method for the generation of amutein of human tear lipocalin includes mutating at least 2, 3, 4, 5, 6,8, 10, 12, 14, 15, 16, or 17 of the codons of any of the amino acidsequence positions 26-34, 56-58, 80, 83, 104-106, and 108 of the linearpolypeptide sequence of mature human tear lipocalin. In anotherembodiment all 18 of the codons of amino acid sequence positions 26, 27,28, 29, 30, 31, 32, 33, 34, 56, 57, 58, 80, 83, 104, 105, 106, and 108of the linear polypeptide sequence of mature human tear lipocalin aremutated.

In another aspect, the present invention includes a method for thegeneration of a mutein of human tear lipocalin, wherein the mutein bindsa given non-natural ligand of human tear lipocalin with detectablebinding affinity, including:

-   -   (a) subjecting a nucleic acid molecule encoding a human tear        lipocalin to mutagenesis at at least one codon of any of the        amino acid sequence positions 34, 80, and 104 of the linear        polypeptide sequence of mature human tear lipocalin, thereby        obtaining a plurality of nucleic acids encoding muteins of human        tear lipocalin,    -   (b) expressing the one or more mutein nucleic acid molecule(s)        obtained in (a) in an expression system, thereby obtaining one        or more mutein(s), and    -   (c) enriching the one or more mutein(s) obtained in step (b) and        having detectable binding affinity for a given non-natural        ligand of human tear lipocalin by means of selection and/or        isolation.

In one embodiment of the afore-mentioned method, additionally at least2, 3, 4, 5, 6, 8, 10, 12, 14, or 15 of the codons of any of the aminoacid sequence positions 26-33, 56-58, 83, 105-106, and 108 of the linearpolypeptide sequence of mature human tear lipocalin are mutated.

In a further embodiment of the invention, the methods according to theinvention include the mutation of both of the codons encoding cysteineat positions 61 and 153 in the linear polypeptide sequence of maturehuman tear lipocalin. In one embodiment position 61 is mutated to encodean alanine, phenylalanine, lysine, arginine, threonin, asparagine,tyrosine, methionine, serine, proline or a tryptophane residue, to nameonly a few possibilities. In embodiments where position 153 is mutated,an amino acid such as a serine or alanine can be introduced at position153.

In another embodiment of the invention as described herein, the codonsencoding amino acid sequence positions 111 and/or 114 of the linearpolypeptide sequence of mature human tear lipocalin are mutated toencode for example an arginine at position 111 and a tryptophane atposition 114.

Another embodiment of the methods of the invention, involves mutagenesisof the codon encoding the cysteine at position 101 of the linearpolypeptide sequence of mature human tear lipocalin so that this codonencodes any other amino acid. In one embodiment the mutated codonencoding position 101 encodes a serine. Accordingly, in some embodimentseither two or all three of the cystein codons at position 61, 101 and153 are replaced by a codon of another amino acid.

According to the method of the invention a mutein is obtained startingfrom a nucleic acid encoding human tear lipocalin. Such a nucleic acidis subjected to mutagenesis and introduced into a suitable bacterial oreukaryotic host organism by means of recombinant DNA technology.Obtaining a nucleic acid library of tear lipocalin can be carried outusing any suitable technique that is known in the art for generatinglipocalin muteins with antibody-like properties, i.e. muteins that haveaffinity towards a given target. Examples of such combinatorial methodsare described in detail in the international patent applications WO99/16873, WO 00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO2005/019254, WO 2005/019255, WO 2005/019256, or WO 2006/56464 forinstance. The content of each of these patent applications isincorporated by reference herein in its entirety. After expression ofthe nucleic acid sequences that were subjected to mutagenesis in anappropriate host, the clones carrying the genetic information for theplurality of respective lipocalin muteins, which bind a given target canbe selected from the library obtained. Well known techniques can beemployed for the selection of these clones, such as phage display(reviewed in Kay, B. K. et al. (1996) supra; Lowman, H. B. (1997) supraor Rodi, D. J., and Makowski, L. (1999) supra), colony screening(reviewed in Pini, A. et al. (2002) Comb. Chem. High Throughput Screen.5, 503-510), ribosome display (reviewed in Amstutz, P. et al. (2001)Curr. Opin. Biotechnol. 12, 400-405) or mRNA display as reported inWilson, D. S. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755 orthe methods specifically described in WO 99/16873, WO 00/75308, WO03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255,WO 2005/019256, or WO 2006/56464.

In accordance with this disclosure, step (c) further comprises inanother embodiment of the above methods:

-   -   (i) providing as a given ligand a compound selected from the        group consisting of a chemical compound in free or conjugated        form that exhibits features of an immunological hapten, a        peptide, a protein or another macromolecule such as a        polysaccharide, a nucleic acid molecule (DNA or RNA, for        example) or an entire virus particle or viroid, for example,    -   (ii) contacting the plurality of muteins with said ligand in        order to allow formation of complexes between said ligand and        muteins having binding affinity for said ligand, and    -   (iii) removing muteins having no or no substantial binding        affinity.

In some embodiments of the invention, the ligand may be a protein or afragment thereof. In one of these embodiments muteins binding the humanT-cell coreceptor CD4 are excluded.

In one embodiment of the methods of the invention, the selection in step(c) is carried out under competitive conditions. Competitive conditionsas used herein means that selection of muteins encompasses at least onestep in which the muteins and the given non-natural ligand of human tearlipocalin (target) are brought in contact in the presence of anadditional ligand, which competes with binding of the muteins to thetarget. This additional ligand may be a physiological ligand of thetarget, an excess of the target itself or any other non-physiologicalligand of the target that binds at least an overlapping epitope to theepitope recognized by the muteins of the invention and thus interfereswith target binding of the muteins. Alternatively, the additional ligandcompetes with binding of the muteins by complexing an epitope distinctfrom the binding site of the muteins to the target by allostericeffects.

An embodiment of the phage display technique (reviewed in Kay, B. K. etal. (1996), supra; Lowman, H. B. (1997) supra or Rodi, D. J., andMakowski, L. (1999), supra) using temperent M13 phage is given as anexample of a selection method that can be employed in the presentinvention. Another embodiment of the phage display technology that canbe used for selection of muteins of the invention is the hyperphagephage technology as described by Broders et al. (Broders et al. (2003)“Hyperphage. Improving antibody presentation in phage display.” MethodsMol. Biol. 205:295-302). Other temperent phage such as fl or lytic phagesuch as T7 may be employed as well. For the exemplary selection method,M13 phagemids are produced which allow the expression of the mutatedlipocalin nucleic acid sequence as a fusion protein with a signalsequence at the N-terminus, preferably the OmpA-signal sequence, andwith the capsid protein pIII of the phage M13 or fragments thereofcapable of being incorporated into the phage capsid at the C-terminus.The C-terminal fragment ΔpIII of the phage capsid protein comprisingamino acids 217 to 406 of the wild type sequence is preferably used toproduce the fusion proteins. Especially preferred in one embodiment is aC-terminal fragment of pIII, in which the cysteine residue at position201 is missing or is replaced by another amino acid.

Accordingly, a further embodiment of the methods of the inventioninvolves operably fusing a nucleic acid coding for the plurality ofmuteins of human tear lipocalin and resulting from mutagenesis at the 3′end with a gene coding for the coat protein pIII of a filamentousbacteriophage of the M13-family or for a fragment of this coat protein,in order to select at least one mutein for the binding of a givenligand.

The fusion protein may comprise additional components such as anaffinity tag, which allows the immobilization, detection and/orpurification of the fusion protein or its parts. Furthermore, a stopcodon can be located between the sequence regions encoding the lipocalinor its muteins and the phage capsid gene or fragments thereof, whereinthe stop codon, preferably an amber stop codon, is at least partiallytranslated into an amino acid during translation in a suitablesuppressor strain.

For example, the phasmid vector pTLPC27, now also called pTlc27 that isdescribed here can be used for the preparation of a phagemid libraryencoding human tear lipocalin muteins. The inventive nucleic acidmolecules coding for the tear lipocalin muteins are inserted into thevector using the two BstXI restriction sites. After ligation a suitablehost strain such as E. coli XL1-Blue is transformed with the resultingnucleic acid mixture to yield a large number of independent clones. Arespective vector can be generated for the preparation of ahyperphagemid library, if desired.

The resulting library is subsequently superinfected in liquid culturewith an appropriate M13-helper phage or hyperphage in order to producefunctional phagemids. The recombinant phagemid displays the lipocalinmutein on its surface as a fusion with the coat protein pIII or afragment thereof, while the N-terminal signal sequence of the fusionprotein is normally cleaved off. On the other hand, it also bears one ormore copies of the native capsid protein pIII supplied by the helperphage and is thus capable of infecting a recipient, in general abacterial strain carrying an F- or F′-plasmid. In case of hyperphagedisplay, the hyperphagemids display the lipocalin muteins on theirsurface as a fusion with the infective coat protein pIII but no nativecapsid protein. During or after infection with helper phage orhyperphage, gene expression of the fusion protein between the lipocalinmutein and the capsid protein pIII can be induced, for example byaddition of anhydrotetracycline. The induction conditions are chosensuch that a substantial fraction of the phagemids obtained displays atleast one lipocalin mutein on their surface. In case of hyperphagedisplay induction conditions result in a population of hyperphagemidscarrying between three and five fusion proteins consisting of thelipocalin mutein and the capsid protein pIII. Various methods are knownfor isolating the phagemids, such as precipitation with polyethyleneglycol. Isolation typically occurs after an incubation period of 6-8hours.

The isolated phasmids can then be subjected to selection by incubationwith the desired target, wherein the target is presented in a formallowing at least temporary immobilization of those phagemids whichcarry muteins with the desired binding activity as fusion proteins intheir coat. Among the various embodiments known to the person skilled inthe art, the target can, for example, be conjugated with a carrierprotein such as serum albumin and be bound via this carrier protein to aprotein binding surface, for example polystyrene. Microtiter platessuitable for ELISA techniques or so-called “immuno-sticks” canpreferrably be used for such an immobilization of the target.Alternatively, conjugates of the target with other binding groups, suchas biotin, can be used. The target can then be immobilized on a surfacewhich selectively binds this group, for example microtiter plates orparamagnetic particles coated with streptavidin, neutravidin or avidin.If the target is fused to an Fc portion of an immunoglobulin,immobilization can also be achieved with surfaces, for examplemicrotiter plates or paramagnetic particles, which are coated withprotein A or protein G.

Non-specific phagemid-binding sites present on the surfaces can besaturated with blocking solutions as they are known for ELISA methods.The phagemids are then typically brought into contact with the targetimmobilized on the surface in the presence of a physiological buffer.Unbound phagemids are removed by multiple washings. The phagemidparticles remaining on the surface are then eluted. For elution, severalmethods are possible. For example, the phagemids can be eluted byaddition of proteases or in the presence of acids, bases, detergents orchaotropic salts or under moderately denaturing conditions. A preferredmethod is the elution using buffers of pH 2.2, wherein the eluate issubsequently neutralized. Alternatively, a solution of the free targetcan be added in order to compete with the immobilzed target for bindingto the phagemids or target-specific phagemids can be eluted bycompetition with immunoglobulins or natural liganding proteins whichspecifically bind to the target of interest.

Afterwards, E. coli cells are infected with the eluted phagemids.Alternatively, the nucleic acids can be extracted from the elutedphagemids and used for sequence analysis, amplification ortransformation of cells in another manner. Starting from the E. coliclones obtained in this way, fresh phagemids or hyperphagemids are againproduced by superinfection with M13 helper phages or hyperphageaccording to the method described above and the phagemids amplified inthis way are once again subjected to a selection on the immobilizedtarget. Multiple selection cycles are often necessary in order to obtainthe phagemids with the muteins of the invention in sufficiently enrichedform. The number of selection cycles is preferably chosen such that inthe subsequent functional analysis at least 0.1% of the clones studiedproduce muteins with detectable affinity for the given target. Dependingon the size, i.e. the complexity of the library employed, 2 to 8 cyclesare typically required to this end.

For the functional analysis of the selected muteins, an E. coli strainis infected with the phagemids obtained from the selection cycles andthe corresponding double stranded phasmid DNA is isolated. Starting fromthis phasmid DNA, or also from the single-stranded DNA extracted fromthe phagemids, the nucleic acid sequences of the selected muteins of theinvention can be determined by the methods known in the art and theamino acid sequence can be deduced therefrom. The mutated region or thesequence of the entire tear lipocalin mutein can be subcloned on anotherexpression vector and expressed in a suitable host organism. Forexample, the vector pTLPC26 now also called pTlc26 can be used forexpression in E. coli strains such as E. coli TG1. The muteins of tearlipocalin thus produced can be purified by various biochemical methods.The tear lipocalin muteins produced, for example with pTlc26, carry theaffinity peptide Strep-tag II (Schmidt et al., supra) at their C-terminiand can therefore preferably be purified by streptavidin affinitychromatography.

The selection can also be carried out by means of other methods. Manycorresponding embodiments are known to the person skilled in the art orare described in the literature. Moreover, a combination of methods canbe applied. For example, clones selected or at least enriched by “phagedisplay” can additionally be subjected to “colony screening”. Thisprocedure has the advantage that individual clones can directly beisolated with respect to the production of a tear lipocalin mutein withdetectable binding affinity for a target.

In addition to the use of E. coli as host organism in the “phagedisplay” technique or the “colony screening” method, other bacterialstrains, yeast or also insect cells or mammalian cells can be used forthis purpose. Further to the selection of a tear lipocalin mutein from arandom library as described above, evolutive methods including limitedmutagenesis can also be applied in order to optimize a mutein thatalready possesses some binding activity for the target with respect toaffinity or specificity for the target after repeated screening cycles.

Once a mutein with affinity to a given target has been selected, it isadditionally possible to subject such a mutein to another mutagenesis inorder to subsequently select variants of even higher affinity orvariants with improved properties such as higher thermostability,improved serum stability, thermodynamic stability, improved solubility,improved monomeric behavior, improved resistance against thermaldenaturation, chemical denaturation, proteolysis, or detergents etc.This further mutagenesis, which in case of aiming at higher affinity canbe considered as in vitro “affinity maturation”, can be achieved by sitespecific mutation based on rational design or a random mutation. Anotherpossible approach for obtaining a higher affinity or improved propertiesis the use of error-prone PCR, which results in point mutations over aselected range of sequence positions of the lipocalin mutein. Theerror-prone PCR can be carried out in accordance with any known protocolsuch as the one described by Zaccolo et al. (1996) J. Mol. Biol. 255,589-603. Other methods of random mutagenesis that are suitable for suchpurposes include random insertion/deletion (RID) mutagenesis asdescribed by Murakami, H et al. (2002) Nat. Biotechnol. 20, 76-81 ornonhomologous random recombination (NRR) as described by Bittker, J. Aet al. (2002) Nat. Biotechnol. 20, 1024-1029. If desired, affinitymaturation can also be carried out according to the procedure describedin WO 00/75308 or Schlehuber, S. et al., (2000) J. Mol. Biol. 297,1105-1120, where muteins of the bilin-binding protein having highaffinity to digoxigenin were obtained.

In a further aspect, the present invention is directed to a mutein ofhuman tear lipocalin having detectable binding affinity to a givennon-natural ligand of human tear lipocalin, which is obtainable by orobtained by the above-detailed methods of the invention.

In one embodiment, the mutein of human tear lipocalin obtained accordingto the above methods includes the substitution of at least one or ofboth of the cysteine residues occurring at each of the sequencespositions 61 and 153 by another amino acid and the mutation of at leastone amino acid residue at any one of the sequence positions 26-34,56-58, 80, 83, 104-106, and 108 of the linear polypeptide sequence ofmature human tear lipocalin. The positions 24-36 are comprised in the ABloop, the positions 53-66 are comprised in the CD loop, the positions69-77 are comprised in the EF loop and the positions 103-110 arecomprised in the GH loop in the binding site at the open end of theβ-barrel structure of tear lipocalin. The definition of these four loopsis used herein in accordance with Flower (Flower, D. R. (1996), supraand Flower, D. R. et al. (2000), supra). Usually, such a muteincomprises at least 2, 3, 4, 5, 6, 8, 10, 12, 14, 15, 16, 17 or 18mutated amino acid residues at the sequence positions 26-34, 56-58, 80,83, 104-106, and 108 of the linear polypeptide sequence of mature humantear lipocalin. In a specific embodiment, the mutein comprises the aminoacid substitutions Cys 61→Ala, Phe, Lys, Arg, Thr, Asn, Tyr, Met, Ser,Pro or Trp and Cys 153→Ser or Ala. Such a substitution has proven usefulto prevent the formation of the naturally occurring disulphide bridgelinking Cys 61 and Cys 153, and thus to facilitate handling of themutein.

In still another embodiment, the mutein comprises at least oneadditional amino acid substitution selected from Arg 111→Pro and Lys114→Trp. A mutein of the invention may further comprise the cysteine atposition 101 of the sequence of native mature human tear lipocalinsubstituted by another amino acid. This substitution may, for example,be the mutation Cys 101→Ser or Cys 101→Thr.

The non-natural ligand the mutein is binding to may be protein or afragment thereof with the proviso that in some embodiments the humanT-cell coreceptor CD4 may be excluded as non natural target.

The lipocalin muteins of the invention may comprise the wild type(natural) amino acid sequence outside the mutated amino acid sequencepositions. On the other hand, the lipocalin muteins disclosed herein mayalso contain amino acid mutations outside the sequence positionssubjected to mutagenesis as long as those mutations do not interferewith the binding activity and the folding of the mutein. Such mutationscan be accomplished very easily on DNA level using established standardmethods (Sambrook, J. et al. (1989) Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Possible alterations of the amino acid sequence areinsertions or deletions as well as amino acid substitutions. Suchsubstitutions may be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. One the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of tear lipocalin as long as these deletions orinsertion result in a stable folded/functional mutein (see for example,the experimental section in which muteins with truncated N- andC-terminus are generated).

Such modifications of the amino acid sequence include directedmutagenesis of single amino acid positions in order to simplifysub-cloning of the mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for a given target. Furthermore, mutations can beintroduced in order to modulate certain characteristics of the muteinsuch as to improve folding stability, serum stability, proteinresistance or water solubility or to reduce aggregation tendency, ifnecessary. For example, naturally occurring cysteine residues may bemutated to other amino acids to prevent disulphide bridge formation.However, it is also possible to deliberately mutate other amino acidsequence position to cysteine in order to introduce new reactive groups,for example for the conjugation to other compounds, such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins,or for the formation of non-naturally occurring disulphide linkages.Exemplary possibilities of such a mutation to introduce a cysteineresidue into the amino acid sequence of a human tear lipocalin muteininclude the substitutions Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp95→Cys, and Glu 131→Cys. The generated thiol moiety at the side of anyof the amino acid positions 40, 73, 90, 95 and/or 131 may be used toPEGylate or HESylate the mutein, for example, in order to increase theserum half-life of a respective tear lipocalin mutein. The muteinS236.1-A22 into which a cysteine is introduced at any these sequencepositions (see Example 46) is an illustrative example of such muteins ofthe invention.

The present invention also encompasses muteins as defined above, inwhich the first four N-terminal amino acid residues of the sequence ofmature human tear lipocalin (His-His-Leu-Leu; positions 1-4) and/or thelast two C-terminal amino acid residues (Ser-Asp; positions 157-158) ofthe sequence of mature human tear lipocalin have been deleted (cf. alsothe Examples and the attached Sequence Listings).

The lipocalin muteins of the invention are able to bind the desiredtarget with detectable affinity, i.e. with a dissociation constant of atleast 200 nM. Presently preferred in some embodiments are lipocalinmuteins, which bind the desired target with a dissociation constant fora given target of at least 100, 20, 1 nM or even less. The bindingaffinity of a mutein to the desired target can be measured by amultitude of methods such as fluorescence titration, competition ELISAor surface plasmon resonance (BIAcore).

It is readily apparent to the skilled person that complex formation isdependent on many factors such as concentration of the binding partners,the presence of competitors, ionic strength of the buffer system etc.Selection and enrichment is generally performed under conditionsallowing the isolation of lipocalin muteins having, in complex with thedesired target, a dissociation constant of at least 200 nM. However, thewashing and elution steps can be carried out under varying stringency. Aselection with respect to the kinetic characteristics is possible aswell. For example, the selection can be performed under conditions,which favor complex formation of the target with muteins that show aslow dissociation from the target, or in other words a low k_(off) rate.Alternatively, selection can be perfomed under conditions, which favourfast formation of the complex between the mutein and the target, or inother words a high k_(on) rate. As a further illustrative alternative,the screening can be performed under conditions that select for improvedthermostability of the muteins (compared to either wild type tearlipocalin or a mutein that already has affinity towards a pre-selectedtarget)

A tear lipocalin mutein of the invention typically exists as monomericprotein. However, it is also possible that an inventive lipocalin muteinis able to spontaneously dimerise or oligomerise. Although the use oflipocalin muteins that form stable monomers may be preferred for someapplications, e.g. because of faster diffusion and better tissuepenetration, the use of lipocalin muteins that spontaneously form stablehomodimers or multimers may be advantageous in other instances, sincesuch multimers can provide for a (further) increased affinity and/oravidity to a given target. Furthermore, oligomeric forms of thelipocalin mutein may have slower dissociation rates or prolonged serumhalf-life. If dimerisation or multimerisation of muteins that formstable monomers is desired, this can for example be achieved by fusingrespective oligomerization domains such as jun-fos domains orleucin-zippers to muteins of the invention or by the use of “Duocalins”(see also below).

A tear lipocalin mutein of the invention may be used for complexformation with a given target. The target may be anon-naturaltarget/ligand. The target (ligand) may be any chemical compound in freeor conjugated form which exhibits features of an immunological hapten, ahormone such as steroid hormones or any biopolymer or fragment thereof,for example, a protein or protein domain, a peptide, anoligodeoxynucleotide, a nucleic acid, an oligo- or polysaccharide orconjugates thereof. In one embodiment of the invention the target is aprotein with the proviso that the human T-cell coreceptor CD4 isexcluded. The protein can be any globular soluble protein or a receptorprotein, for example, a trans-membrane protein involved in cellsignaling, a component of the immune systems such as an MHC molecule orcell surface receptor that is indicative of a specific disease. Themutein may also be able to bind only fragments of a protein. Forexample, a mutein can bind to a domain of a cell surface receptor, whenit is part of the receptor anchored in the cell membrane as well as tothe same domain in solution, if this domain can be produced as a solubleprotein as well. However the invention is by no means limited to muteinsthat only bind such macromolecular targets. But it is also possible toobtain muteins of tear lipocalin by means of mutagenesis which showspecific binding affinity to ligands of low(er) molecular weight such asbiotin, fluorescein or digoxigenin.

In one embodiment of the invention the ligand that is bound by the tearlipocalin mutein is a protein or fragment thereof selected from thegroup of vascular endothelial growth factor (VEGF), vascular endothelialgrowth factor receptor 2 (VEGF-R2), and interleukin 4 receptor alphachain (IL-4 receptor alpha) or fragments thereof. Also included asligands are an extracellular region or a domain of VEGF-R2 or IL-4receptor alpha. These ligands are typically of mammalian origin. In oneembodiment these ligands are of human origin, but they may also be ofmouse, rat, porcine, equine, canine, feline or bovine or cynomolgusorigin, to name only a few illustrative examples.

Human VEGF may be selected from the group consisting of VEGF-A, VEGF-B,VEGF-C, and VEGF-D and may have the amino acid sequences set forth inSWISS PROT Data Bank Accession Nos. P15692, P49765, P49767, and 043915(SEQ ID Nos.: 22-25) or of fragments thereof. One such exemplaryfragment consists of amino acids 8 to 109 of VEGF-A. Human vascularendothelial groth factor receptor 2 (VEGF-R2) may have the amino acidsequence of SWISS PROT Data Bank Accession No. P35968 (SEQ ID NO: 21) orof fragments thereof. Illustrative examples of such fragments includethe extracellular Ig-like C2-type domains 1 to 7 of VEGF-R2, comprisingamino acids 46 to 110, 141 to 207, 224 to 320, 328 to 414, 421 to 548,551 to 660, and 667 to 753, respectively. Human interleukin-4 receptoralpha chain may have the amino acid sequence of SWISS PROT Data BankAccession No. P24394 (SEQ ID NO: 20) or of fragments thereof. Anillustrative example of a fragment of human interleukin-4 receptor alphachain includes amino acids 26 to 232 of IL-4 receptor alpha.

In general, the term “fragment”, as used herein with respect to proteinligands of the tear lipocalin muteins of the invention, relates toN-terminally and/or C-terminally shortened protein or peptide ligands,which retain the capability of the full length ligand to be recognizedand/or bound by a mutein according to the invention.

Therefore, another aspect of the present invention is directed to amutein of human tear lipocalin that comprises at least one mutated aminoacid residue at any two or more of the sequence positions 24-36, 53-66,79-84, and 103-110 of the linear polypeptide sequence of the maturehuman tear lipocalin, and binds to IL-4 receptor alpha, VEGF-R2 or VEGF.

Human tear lipocalin muteins binding IL-4 receptor alpha may act as IL-4antagonists and/or IL-13 antagonists. In one embodiment, the human tearlipocalin muteins act as antagonists of human IL-4 and/or IL-13. Inanother embodiment, the mutein is cross-reactive with the cynomolgusligands such as IL-4 and/or IL-13 and as such acts as an antagonist ofcynomolgus IL-4 receptor alpha.

A human tear lipocalin mutein of the invention that binds IL-4 receptoralpha may comprise with respect to the amino acid sequence of maturehuman tear lipocalin at least two amino acid substitutions of nativeamino acid residues by cysteine residues at any of positions 26-34,56-58, 80, 83, 104-106, and 108 of native mature human tear lipocalin.Generally, such a mutein binds an extracellular region or a domain ofIL-4 receptor alpha with a K_(D) of 200 nM or less, 100 nM or less, 20nM or less, or 1 nM or even less with a K_(D) in the picomolar range.Thus, the invention also encompasses tear lipocalin muteins that bindIL-4 receptor with a K_(D) of 900 pM or less, 600 pM or less, 500 pM orless, 250 pM, 100 pM or less, 60 pM or less or 40 pM or less. Suitablemethods to determine K_(D) values of a mutein-ligand complex are knownto those skilled in the art and include fluorescence titration,competition ELISA, calorimetric methods, such as isothermal titrationcalorimetry (ITC), and surface plasmon resonance. Examples for suchmethods are detailed below (See, e.g., Examples 6, 8, 14, 16, 22, 24,and 27).

In this context it is also noted that the complex formation between therespective mutein and its ligand is influenced by many different factorssuch as the concentrations of the respective binding partners, thepresence of competitors, pH and the ionic strength of the buffer systemused, and the experimental method used for determination of thedissociation constant K_(D) (for example fluorescence titration,competition ELISA or surface plasmon resonance, just to name a few) oreven the mathematical algorithm which is used for evaluation of theexperimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivemutein and its ligand) given here may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means, there may be a slight deviation in the measuredK_(D) values or a tolerance range depending, for example, on whether theK_(D) value was determined by surface plasmon resonance (Biacore) or bycompetition ELISA.

In a specific embodiment of the invention such a mutein comprises withrespect to the amino acid sequence of mature human tear lipocalin atleast 6, 8, 10, 12, 14 or 16 amino acid substitutions selected from thegroup consisting of Arg 26→Ser, Pro; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg;Met 31→Ala; Asn 32→Tyr, His; Leu 33→Tyr; Glu 34→Gly, Ser, Ala, Asp, Lys,Asn, Thr, Arg; Leu 56→Gln; Ile 57→Arg; Ser 58→Ile, Ala, Arg, Val, Thr,Asn, Lys, Tyr, Leu, Met; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu105→Cys; His 106→Pro; and Lys 108→Gln.

Additionally, such a mutein may further comprise at least one amino acidsubstitution selected from the group consisting of Met 39→Val; Thr42→Met, Ala; Thr 43→Ile, Pro, Ala; Glu 45→Lys, Gly; Asn 48→Asp, His,Ser, Thr; Val 53→Leu, Phe, Ile, Ala, Gly, Ser; Thr 54→Ala, Leu; Met55→Leu, Ala, Ile, Val, Phe, Gly, Thr, Tyr; Glu 63→Lys, Gln, Ala, Gly,Arg; Val 64→Gly, Tyr, Met, Ser, Ala, Lys, Arg, Leu, Asn, His, Thr, Ile;Ala 66→Ile, Leu, Val, Thr, Met; Glu 69→Lys, Gly; Lys 70→Arg, Gln, Glu;Thr 78→Ala; Ile 89→Val; Asp 95→Asn, Ala, Gly; and Tyr 100→His.

In one embodiment, the human tear lipocalin mutein binding IL-4 receptoralpha comprises the amino acid substitutions: Arg 26→Ser, Glu 27→Arg,Phe 28→Cys, Glu 30→Arg; Met 31→Ala, Leu 33→Tyr, Leu 56→Gln, Ile 57→Arg,Asp 80→Ser, Lys 83→Arg, Glu 104→Leu, Leu 105→Cys, His 106→Pro, and Lys108→Gln.

In another embodiment, the human tear lipocalin mutein binding IL-4receptor alpha comprises one of the following sets of amino acidsubstitutions:

-   -   (1) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Glu 34→Gly; Leu 56→Gln; Ile 57→Arg; Ser        58→Ile; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His        106→Pro; Lys 108→Gln;    -   (2) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Glu 34→Lys; Leu 56→Gln; Ile 57→Arg; Ser        58→Asn; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His        106→Pro; Lys 108→Gln;    -   (3) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys, Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Leu 56→Gln; Ile 57→Arg; Ser 58→Arg; Asp        80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His 106→Pro; Lys        108→Gln;    -   (4) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Glu 34→Ser; Leu 56→Gln; Ile 57→Arg; Asp        80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His 106→Pro; Lys        108→Gln;    -   (5) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→His; Leu 33→Tyr; Glu 34→Ser; Leu 56→Gln; Ile 57→Arg; Ser        58→Ala; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His        106→Pro; Lys 108→Gln;    -   (6) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Glu 34→Asp; Leu 56→Gln; Ile 57→Arg; Ser        58→Lys; Asp 80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His        106→Pro; Lys 108→Gln; and    -   (7) Arg 26→Ser; Glu 27→Arg; Phe 28→Cys; Glu 30→Arg; Met 31→Ala;        Asn 32→Tyr; Leu 33→Tyr; Glu 34→Gly; Leu 56→Gln; Ile 57→Arg; Asp        80→Ser; Lys 83→Arg; Glu 104→Leu; Leu 105→Cys; His 106→Pro; Lys        108→Gln.

The human tear lipocalin mutein binding IL-4 receptor alpha maycomprise, consists essentially of or consist of any one of the aminoacid sequences set forth in SEQ ID NOs.: 2-8 or a fragment or variantthereof. In one embodiment, the mutein according to the inventioncomprises, consists essentially of or consists of the amino acidsequence set forth in SEQ ID NO: 5 or 6 or a fragment or variantthereof.

The term “fragment” as used in the present invention in connection withthe muteins of the invention relates to proteins or peptides derivedfrom full-length mature human tear lipocalin that are N-terminallyand/or C-terminally shortened, i.e. lacking at least one of theN-terminal and/or C-terminal amino acids. Such fragments comprisepreferably at least 10, more preferably 20, most preferably 30 or moreconsecutive amino acids of the primary sequence of mature human tearlipocalin and are usually detectable in an immunoassay of mature humantear lipocalin.

The term “variant” as used in the present invention relates toderivatives of a protein or peptide that comprise modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Preferably, such modifications do not reduce thefunctionality of the protein or peptide. Such variants include proteins,wherein one or more amino acids have been replaced by their respectiveD-stereoisomers or by amino acids other than the naturally occurring 20amino acids, such as, for example, ornithine, hydroxyproline,citrulline, homoserine, hydroxylysine, norvaline. However, suchsubstitutions may also be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan.

In a further aspect, the present invention is directed to a mutein ofhuman tear lipocalin binding to Vascular Endothelial Growth FactorReceptor 2 (VEGF-R2) or an extracellular region or a domain thereof.Usually, such a mutein acts as a VEGF antagonist and binds anextracellular region or a domain of VEGF-R2 with a K_(D) of 200 nM orless, 100 nM or less, 20 nM or less, 15 nM or less, 10 nM or less oreven 1 nM or less.

Such a mutein may comprise with respect to the amino acid sequence ofmature human tear lipocalin at least 6, 8, 10, 12, 14 or 16 amino acidsubstitutions selected from the group consisting of Arg 26→Ser; Glu27→Ile; Glu 30→Ser; Met 31→Gly; Asn 32→Arg; Leu 33→Ile; Glu 34→Tyr; Leu56→Lys, Glu, Ala, Met; Ile 57→Phe; Ser 58→Arg; Asp 80→Ser, Pro; Lys83→Glu, Gly; Glu 104→Leu; Leu 105→Ala; His 106→Val; and Lys 108→Thr andmay further comprise at least one amino acid substitution selected fromthe group consisting of Leu 41→Phe; Glu 63→Lys; Val 64→Met; Asp 72→Gly;Lys 76→Arg, Glu; Ile 88→Val, Thr; Ile 89→Thr; Arg 90→Lys; Asp 95→Gly;Phe 99→Leu; and Gly 107→Arg, Lys, Glu.

In one specific embodiment, such a mutein comprises the amino acidsubstitutions: Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn32→Arg, Leu 33→Ile, Glu 34 Tyr, Ile 57→Phe, Ser 58→Arg, Lys 83→Glu, Glu104→Leu, Leu 105→Ala, His 106→Val, and Lys 108→Thr.

A human tear lipocalin mutein of the invention that binds to anextracellular region or a domain of VEGF-R2 with detectable affinity maycomprise one of the following sets of amino acid substitutions:

-   -   (1) Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg,        Leu 33→Ile, Glu 34→Tyr, Leu 56→Lys, Ile 57→Phe, Ser 58→Arg, Asp        80→Ser, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, Lys        108→Thr;    -   (2) Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg,        Leu 33→Ile, Glu 34→Tyr, Leu 56→Glu, Ile 57→Phe, Ser 58→Arg, Asp        80→Ser, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, Lys        108→Thr;    -   (3) Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg,        Leu 33→Ile, Glu 34→Tyr, Leu 56→Ala, Ile 57→Phe, Ser 58→Arg, Asp        80→Ser, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, Lys        108→Thr; and    -   (4) Arg 26→Ser, Glu 27→Ile, Glu 30→Ser, Met 31→Gly, Asn 32→Arg,        Leu 33→Ile, Glu 34→Tyr, Leu 56→Glu, Ile 57→Phe, Ser 58→Arg, Asp        80→Pro, Lys 83→Glu, Glu 104→Leu, Leu 105→Ala, His 106→Val, Lys        108→Thr.

In one embodiment of the invention, the mutein binding to VEGF-R2comprises, consists essentially of or consists of any one of the aminoacid sequences set forth in SEQ ID Nos.: 34-39.

In a still further aspect, the present invention is directed to a muteinof human tear lipocalin binding to Vascular Endothelial Growth Factor(VEGF). Usually, such a mutein acts a a VEGF antagonist by inhibitingthe binding of VEGF to the VEGF receptor and binds VEGF with a K_(D) of200 nM or less, 100 nM or less, 20 nM, 5 nM or less or even 1 nM orless.

Such a mutein obtainable by the methods of the invention may comprisewith respect to the amino acid sequence of mature human tear lipocalinat least 6, 8, 10, 12, 14, 16 amino acid substitutions selected from thegroup consisting of Arg 26→Ser, Pro, Val, Leu, Ile; Glu 27→Gly; Phe28→Ala; Pro 29→Leu; Glu 30→Arg; Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu34→Gly; Leu 56→His, Arg, Tyr, Gln; Ile 57→Val, Thr, Leu; Ser 58→Lys; Asp80→Ile; Lys 83→Ile, Val; Glu 104→Cys; His 106→Asn, Ser, Asp; and Lys108→Ala, Val and may further comprise at least one amino acidsubstitution selected from the group consisting of Val 36→Met; Thr37→Ala; Met 39→Thr; Thr 40→Ala, Ser; Asn 48→Asp; Ala 51→Val; Lys 52→Arg;Thr 54→Val; Met 55→Val; Ser 61→Pro; Lys 65→Arg; Ala 66→Val; Val 67→Ile;Glu 69→Gly, Ser, Thr; Lys 76→Arg, Ile, Ala, Met, Pro; Tyr 87→Arg, His,Lys, Gln; Ile 89→Thr, Val, Gly, His, Met, Lys; Arg 90→Gly; Ile 98→Val;and Gly 107→Glu.

In one embodiment, such a mutein of human tear lipocalin that binds VEGFcomprises the amino acid substitutions: Glu 27→Gly, Phe 28→Ala, Pro29→Leu, Glu 30→Arg, Met 31→Cys, Asn 32→Leu, Leu 33→Ala, Glu 34→Gly, Asp80→Ile, Lys 83→Ile, Glu 104→Cys, and Lys 108→Val.

In another specific embodiment, the mutein of human tear lipocalin thatbinds VEGF may comprise one of the following sets of amino acidsubstitutions:

-   -   (1) Arg 26→Ser; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Asn; Lys        108→Val;    -   (2) Arg 26→Pro; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Glu; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val;    -   (3) Arg 26→Pro; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Asn; Lys        108→Val;    -   (4) Arg 26→Pro; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→Arg; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val;    -   (5) Arg 26→Pro; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val;    -   (6) Arg 26→Ser; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val;    -   (7) Arg 26→Val; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val;    -   (8) Arg 26→Leu; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val; and    -   (9) Arg 26→Ile; Glu 27→Gly; Phe 28→Ala; Pro 29→Leu; Glu 30→Arg;        Met 31→Cys; Asn 32→Leu; Leu 33→Ala; Glu 34→Gly; Leu 56→His; Ser        58→Lys; Asp 80→Ile; Lys 83→Ile; Glu 104→Cys; His 106→Ser; Lys        108→Val.

In one embodiment of the invention, the mutein binding to VEGFcomprises, consists essentially of or consists of any one of the aminoacid sequences set forth in SEQ ID Nos.: 26-33 or SEQ ID Nos.: 44-47.

Also included in the scope of the present invention are the abovemuteins, which have been altered with respect to their potentialimmunogenicity.

Cytotoxic T-cells recognize peptide antigens on the cell surface of anantigen-presenting cell in association with a class I majorhistocompatibility complex (MHC) molecule. The ability of the peptidesto bind to MHC molecules is allele specific and correlates with theirimmunogenicity. In order to reduce immunogenicity of a given protein,the ability to predict which peptides in a protein have the potential tobind to a given MHC molecule is of great value. Approaches that employ acomputational threading approach to identify potential T-cell epitopeshave been previously described to predict the binding of a given peptidesequence to MHC class I molecules (Altuvia et al. (1995) J. Mol. Biol.249: 244-250).

Such an approach may also be utilized to identify potential T-cellepitopes in the muteins of the invention and to make depending on itsintended use a selection of a specific mutein on the basis of itspredicted immunogenicity. It may be furthermore possible to subjectpeptide regions which have been predicted to contain T-cell epitopes toadditional mutagenesis to reduce or eliminate these T-cell epitopes andthus minimize immunogenicity. The removal of amphipathic epitopes fromgenetically engineered antibodies has been described (Mateo et al.(2000) Hybridoma 19(6):463-471) and may be adapted to the muteins of thepresent invention.

The muteins thus obtained may possess a minimized immunogenicity, whichis desirable for their use in therapeutic and diagnostic applications,such as those described below.

For some applications, it is also useful to employ the muteins of theinvention in a labeled form. Accordingly, the invention is also directedto lipocalin muteins which are conjugated to a label selected from thegroup consisting of enzyme labels, radioactive labels, colored labels,fluorescent labels, chromogenic labels, luminescent labels, haptens,digoxigenin, biotin, metal complexes, metals, and colloidal gold. Themutein may also be conjugated to an organic molecule. The term “organicmolecule” as used herein preferably denotes an organic moleculecomprising at least two carbon atoms, but preferably not more than 7 or12 rotatable carbon bonds, having a molecular weight in the rangebetween 100 and 2000 Dalton, preferably between 100 and 1000 Dalton, andoptionally including one or two metal atoms.

In general, it is possible to label the lipocalin mutein with anyappropriate chemical substance or enzyme, which directly or indirectlygenerates a detectable compound or signal in a chemical, physical,optical, or enzymatic reaction. An example for a physical reaction andat the same time optical reaction/marker is the emission of fluorescenceupon irradiation or the emission of X-rays when using a radioactivelabel. Alkaline phosphatase, horseradish peroxidase or β-galactosidaseare examples of enzyme labels (and at the same time optical labels)which catalyze the formation of chromogenic reaction products. Ingeneral, all labels commonly used for antibodies (except thoseexclusively used with the sugar moiety in the Fc part ofimmunoglobulins) can also be used for conjugation to the muteins of thepresent invention. The muteins of the invention may also be conjugatedwith any suitable therapeutically active agent, e.g., for the targeteddelivery of such agents to a given cell, tissue or organ or for theselective targeting of cells, e.g., of tumor cells without affecting thesurrounding normal cells. Examples of such therapeutically active agentsinclude radionuclides, toxins, small organic molecules, and therapeuticpeptides (such as peptides acting as agonists/antagonists of a cellsurface receptor or peptides competing for a protein binding site on agiven cellular target). The lipocalin muteins of the invention may,however, also be conjugated with therapeutically active nucleic acidssuch as antisense nucleic acid molecules, small interfering RNAs, microRNAs or ribozymes. Such conjugates can be produced by methods well knownin the art.

In one embodiment, the muteins of the invention may also be coupled to atargeting moiety that targets a specific body region in order to deliverthe inventive muteins to a desired region or area within the body. Oneexample wherein such modification may be desirable is the crossing ofthe blood-brain-barrier. In order to cross the blood-brain barrier, themuteins of the invention may be coupled to moieties that facilitate theactive transport across this barrier (see Gaillard P J, e al.,Diphtheria-toxin receptor-targeted brain drug delivery. InternationalCongress Series. 2005 1277:185-198 or Gaillard P J, e al. Targeteddelivery across the blood-brain barrier. Expert Opin Drug Deliv. 20052(2): 299-309. Such moieties are for example available under the tradename 2B-Trans™ (to-BBB technologies BV, Leiden, NL).

As indicated above, a mutein of the invention may in some embodiments beconjugated to a moiety that extends the serum half-life of the mutein(in this regard see also PCT publication WO 2006/56464 where suchconjugation strategies are described with references to muteins of humanneutrophile gelatinase-associated lipocalin with binding affinity forCTLA-4). The moiety that extends the serum half-life may be apolyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules,such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9),an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, aCH4 domain of an immunoglobulin, albumin or a fragment thereof, analbumin binding peptide, or an albumin binding protein, transferrin toname only a few. The albumin binding protein may be a bacterial albuminbinding protein, an antibody, an antibody fragment including domainantibodies (see U.S. Pat. No. 6,696,245, for example), or a lipocalinmutein with binding activity for albumin. Accordingly, suitableconjugation partners for extending the half-life of a lipocalin muteinof the invention include albumin (Osborn, B. L. et al. (2002)Pharmacokinetic and pharmacodynamic studies of a human serumalbumin-interferon-alpha fusion protein in cynomolgus monkeys J.Pharmacol. Exp. Ther. 303, 540-548), or an albumin binding protein, forexample, a bacterial albumin binding domain, such as the one ofstreptococcal protein G (König, T. and Skerra, A. (1998) Use of analbumin-binding domain for the selective immobilisation of recombinantcapture antibody fragments on ELISA plates. J. Immunol. Methods 218,73-83). Other examples of albumin binding peptides that can be used asconjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis, M. S., Zhang, M., Meng, Y. G., Kadkhodayan, M., Kirchhofer, D.,Combs, D. & Damico, L. A. (2002). “Albumin binding as a general strategyfor improving the pharmacokinetics of proteins.” J. Biol Chem 277,35035-35043).

In other embodiments, albumin itself or a biological active fragment ofalbumin can be used as conjugation partner of a lipocalin mutein of theinvention. The term “albumin” comprises all mammal albumins such ashuman serum albumin or bovine serum albumin or rat albumine. The albuminor fragment thereof can be recombinantly produced as described in U.S.Pat. No. 5,728,553 or European patent applications EP 0 330 451 and EP 0361 991. Recombinant human albumin (Recombumin®) Novozymes Delta Ltd.(Nottingham, UK) can be conjugated or fused to a lipocalin mutein inorder to extend the half-life of the mutein.

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and MA, USA).

Using transferrin as a moiety to extend the serum half-life of themuteins of the invention, the muteins can be genetically fused to the Nor C terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer/half-life extension partner is alsocommercially available from Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the muteins of the invention, the SynFusion™technology, commercially available from Syntonix Pharmaceuticals, Inc(MA, USA), may be used. The use of this Fc-fusion technology allows thecreation of longer-acting biopharmaceuticals and may for example consistof two copies of the mutein linked to the Fc region of an antibody toimprove pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of a mutein of theinvention is to fuse to the N- or C-terminus of a mutein of theinvention long, unstructured, flexible glycine-rich sequences (forexample poly-glycine with about 20 to 80 consecutive glycine residues).This approach disclosed in WO2007/038619, for example, has also beenterm “rPEG” (recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkyleneglycol can be substituted, unsubstituted, linear or branched. It canalso be an activated polyalkylene derivative. Examples of suitablecompounds are polyethylene glycol (PEG) molecules as described in WO99/64016, in U.S. Pat. No. 6,177,074 or in U.S. Pat. No. 6,403,564 inrelation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (see for example, Fuertges et al. (1990) TheClinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins J. Control.Release 11, 139-148). The molecular weight of such a polymer,preferrably polyethylene glycol, may range from about 300 to about70.000 Dalton, including, for example, polyethylene glycol with amolecular weight of about 10.000, of about 20.000, of about 30.000 or ofabout 40.000 Dalton. Moreover, as e.g. described in U.S. Pat. No.6,500,930 or 6,620,413, carbohydrate oligo- and polymers such as starchor hydroxyethyl starch (HES) can be conjugated to a mutein of theinvention for the purpose of serum half-life extension.

If one of the above moieties is conjugated to the human tear lipocalinmutein of the invention, conjugation to an amino acid side chain can beadvantageous. Suitable amino acid side chains may occur naturally in theamino acid sequence of human tear lipocalin or may be introduced bymutagenesis. In case a suitable binding site is introduced viamutagenesis, one possibility is the replacement of an amino acid at theappropriate position by a cysteine residue. In one embodiment, suchmutation includes at least one of Thr 40→Cys, Glu 73→Cys, Arg 90→Cys,Asp 95→Cys or Glu 131→Cys substitution. The newly created cysteineresidue at any of these positions can in the following be utilized toconjugate the mutein to moiety prolonging the serum half-life of themutein, such as PEG or an activated derivative thereof.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above moieties to the muteins of theinvention artificial amino acids may be introduced by mutagenesis.Generally, such artificial amino acids are designed to be more reactiveand thus to facilitate the conjugation to the desired moiety. Oneexample of such an artifical amino acid that may be introduced via anartificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins disclosed herein it may beadvantageous to use them in the form of fusion proteins. In someembodiments, the inventive human tear lipocalin mutein is fused at itsN-terminus or its C-terminus to a protein, a protein domain or a peptidesuch as a signal sequence and/or an affinity tag.

For pharmaceutical applications a mutein of the invention may be fusedto a fusion partner that extends the in vivo serum half-life of themutein (see again PCT publication WO 2006/56464 where suitable fusionpartner are described with references to muteins of human neutrophilegelatinase-associated lipocalin with binding affinity for CTLA-4).Similar to the conjugates described above, the fusion partner may be anFc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4domain of an immunoglubolin, albumin, an albumin binding peptide or analbumin binding protein, to name only a few. Again, the albumin bindingprotein may be a bacterial albumin binding protein or a lipocalin muteinwith binding activity for albumin. Accordingly, suitable fusion partnersfor extending the half-life of a lipocalin mutein of the inventioninclude albumin (Osborn, B. L. et al. (2002) supra J. Pharmacol. Exp.Ther. 303, 540-548), or an albumin binding protein, for example, abacterial albumin binding domain, such as the one of streptococcalprotein G (König, T. and Skerra, A. (1998) supra J. Immunol. Methods218, 73-83). The albumin binding peptides described in Dennis et al,supra (2002) or US patent application 2003/0069395 having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thrcan also be used as fusion partner. It is also possible to use albuminitself or a biological active fragment of albumin as fusion partner of alipocalin mutein of the invention. The term “albumin” comprises allmammal albumins such as human serum albumin or bovine serum albumin orrat serum albumin. The recombinant production of albumin or fragmentsthereof is well known in the art and for example described in U.S. Pat.No. 5,728,553, European patent application EP 0 330 451 or EP 0 361 991.

The fusion partner may confer new characteristics to the inventivelipocalin mutein such as enzymatic activity or binding affinity forother molecules. Examples of suitable fusion proteins are alkalinephosphatase, horseradish peroxidase, gluthation-S-transferase, thealbumin-binding domain of protein G, protein A, antibody fragments,oligomerization domains, lipocalin muteins of same or different bindingspecificity (which results in the formation of “Duocalins”, cf.Schlehuber, S., and Skerra, A. (2001), Duocalins, engineeredligand-binding proteins with dual specificity derived from the lipocalinfold. Biol. Chem. 382, 1335-1342) or toxins.

In particular, it may be possible to fuse a lipocalin mutein of theinvention with a separate enzyme active site such that both “components”of the resulting fusion protein together act on a given therapeutictarget. The binding domain of the lipocalin mutein attaches to thedisease-causing target, allowing the enzyme domain to abolish thebiological function of the target.

Affinity tags such as the Strep-Tag® or Strep-Tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of preferred fusion partners. Finally, proteinswith chromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for a lipocalin mutein of the invention as well.

The term “fusion protein” as used herein also comprises lipocalinmuteins according to the invention containing a signal sequence. Signalsequences at the N-terminus of a polypeptide direct this polypeptide toa specific cellular compartment, for example the periplasm of E. coli orthe endoplasmatic reticulum of eukaryotic cells. A large number ofsignal sequences is known in the art. A preferred signal sequence forsecretion a polypeptide into the periplasm of E. coli is the OmpA-signalsequence.

The present invention also relates to nucleic acid molecules (DNA andRNA) comprising nucleotide sequences coding for muteins as describedherein. Since the degeneracy of the genetic code permits substitutionsof certain codons by other codons specifying the same amino acid, theinvention is not limited to a specific nucleic acid molecule encoding amutein of the invention but includes all nucleic acid moleculescomprising nucleotide sequences encoding a functional mutein.

Therefore, the present invention also includes a nucleic acid sequenceencoding a mutein according to the invention comprising a mutation at atleast one codon of any of the amino acid sequence positions 26-34,56-58, 80, 83, 104-106 and 108 of the linear polypeptide sequence ofnative mature human tear lipocalin, wherein the codons encoding at leastone of the cysteine residues at sequence positions 61 and 153 of thelinear polypeptide sequence of the mature human tear lipocalin have beenmutated to encode any other amino acid residue.

The invention as disclosed herein also includes nucleic acid moleculesencoding tear lipocalin muteins, which comprise additional mutationsoutside the indicated sequence positions of experimental mutagenesis.Such mutations are often tolerated or can even prove to be advantageous,for example if they contribute to an improved folding efficiency, serumstability, thermal stability or ligand binding affinity of the mutein.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it comprises sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions comprise a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the invention can include aregulatory sequence, preferably a promoter sequence. In anotherpreferred embodiment, a nucleic acid molecule of the invention comprisesa promoter sequence and a transcriptional termination sequence. Suitableprokaryotic promoters are, for example, the tet promoter, the lacUV5promoter or the T7 promoter. Examples of promoters useful for expressionin eukaryotic cells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the invention can also be part of a vectoror any other kind of cloning vehicle, such as a plasmid, a phagemid, aphage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is comprised in a phasmid.A phasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or fl, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (reviewed,e.g., in Kay, B. K. et al. (1996) Phage Display of Peptides andProteins—A Laboratory Manual, 1st Ed., Academic Press, New York N.Y.;Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinof the invention, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding lipocalin muteins of the invention, and inparticular a cloning vector containing the coding sequence of such alipocalin mutein can be transformed into a host cell capable ofexpressing the gene. Transformation can be performed using standardtechniques (Sambrook, J. et al. (1989), supra). Thus, the invention isalso directed to a host cell containing a nucleic acid molecule asdisclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of theinvention. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) orprimary mammalian cells

The invention also relates to a method for the production of a mutein ofthe invention, wherein the mutein, a fragment of the mutein or a fusionprotein of the mutein and another polypeptide is produced starting fromthe nucleic acid coding for the mutein by means of genetic engineeringmethods. The method can be carried out in vivo, the mutein can forexample be produced in a bacterial or eucaryotic host organism and thenisolated from this host organism or its culture. It is also possible toproduce a protein in vitro, for example by use of an in vitrotranslation system.

When producing the mutein in vivo a nucleic acid encoding a mutein ofthe invention is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector comprising a nucleic acid molecule encoding a mutein ofthe invention using established standard methods (Sambrook, J. et al.(1989), supra). The host cell is then cultured under conditions, whichallow expression of the heterologous DNA and thus the synthesis of thecorresponding polypeptide. Subsequently, the polypeptide is recoveredeither from the cell or from the cultivation medium.

In some tear lipocalin muteins of the invention, the naturally occurringdisulfide bond between Cys 61 and Cys 153 is removed. Accordingly, suchmuteins (or any other tear lipocalin mutein that does not comprise anintramolecular disulfide bond) can be produced in a cell compartmenthaving a reducing redox milieu, for example, in the cytoplasma ofGram-negative bacteria. In case a lipocalin mutein of the inventioncomprises intramolecular disulfide bonds, it may be preferred to directthe nascent polypeptide to a cell compartment having an oxidizing redoxmilieu using an appropriate signal sequence. Such an oxidizingenvironment may be provided by the periplasm of Gram-negative bacteriasuch as E. coli, in the extracellular milieu of Gram-positive bacteriaor in the lumen of the endoplasmatic reticulum of eukaryotic cells andusually favors the formation of structural disulfide bonds. It is,however, also possible to produce a mutein of the invention in thecytosol of a host cell, preferably E. coli. In this case, thepolypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi M, Seifert C, Hunte C. (2002)“High level production of functional antibody Fab fragments in anoxidizing bacterial cytoplasm.” J. Mol. Biol. 315, 1-8.).

However, a mutein of the invention may not necessarily be generated orproduced only by use of genetic engineering. Rather, a lipocalin muteincan also be obtained by chemical synthesis such as Merrifield solidphase polypeptide synthesis or by in vitro transcription andtranslation. It is for example possible that promising mutations areidentified using molecular modeling and then to synthesize the wanted(designed) polypeptide in vitro and investigate the binding activity fora given target. Methods for the solid phase and/or solution phasesynthesis of proteins are well known in the art (reviewed, e.g., inLloyd-Williams, P. et al. (1997) Chemical Approaches to the Synthesis ofPeptides and Proteins. CRC Press, Boca Raton, Fields, G. B., andColowick, S. P. (1997) Solid Phase Peptide Synthesis. Academic Press,San Diego, or Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5,29-43).

In another embodiment, the muteins of the invention may be produced byin vitro transcription/translation employing well-established methodsknown to those skilled in the art.

The invention also relates to a pharmaceutical composition comprising atleast one inventive mutein of human tear lipocalin or a fusion proteinor conjugate thereof and a pharmaceutically acceptable excipient.

The lipocalin muteins according to the invention can be administered viaany parenteral or non-parenteral (enteral) route that is therapeuticallyeffective for proteinaceous drugs. Parenteral application methodscomprise, for example, intracutaneous, subcutaneous, intramuscular,intratracheal, intranasal, intravitreal or intravenous injection andinfusion techniques, e.g. in the form of injection solutions, infusionsolutions or tinctures, as well as aerosol installation and inhalation,e.g. in the form of aerosol mixtures, sprays or powders. An overviewabout pulmonary drug delivery, i.e. either via inhalation of aerosols(which can also be used in intranasal administration) or intrachealinstiallation is given by J. S. Patton et al. The lungs as a portal ofentry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol. 1pages 338-344, for example). Non-parenteral delivery modes are, forinstance, orally, e.g. in the form of pills, tablets, capsules,solutions or suspensions, or rectally, e.g. in the form ofsuppositories. The muteins of the invention can be administeredsystemically or topically in formulations containing conventionalnon-toxic pharmaceutically acceptable excipients or carriers, additivesand vehicles as desired.

In one embodiment of the present invention the pharmaceutical isadministered parenterally to a mammal, and in particular to humans.Corresponding administration methods include, but are not limited to,for example, intracutaneous, subcutaneous, intramuscular, intratrachealor intravenous injection and infusion techniques, e.g. in the form ofinjection solutions, infusion solutions or tinctures as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. A combination of intravenous and subcutaneousinfusion and/or injection might be most convenient in case of compoundswith a relatively short serum half life. The pharmaceutical compositionmay be an aqueous solution, an oil-in water emulsion or a water-in-oilemulsion.

In this regard it is noted that transdermal delivery technologies, e.g.iontophoresis, sonophoresis or microneedle-enhanced delivery, asdescribed in Meidan V M and Michniak B B 2004 Am. J. Ther. 11(4):312-316, can also be used for transdermal delivery of the muteinsdescribed herein. Non-parenteral delivery modes are, for instance, oral,e.g. in the form of pills, tablets, capsules, solutions or suspensions,or rectal administration, e.g. in the form of suppositories. The muteinsof the invention can be administered systemically or topically informulations containing a variety of conventional non-toxicpharmaceutically acceptable excipients or carriers, additives, andvehicles.

The dosage of the mutein applied may vary within wide limits to achievethe desired preventive effect or therapeutic response. It will, forinstance, depend on the affinity of the compound for a chosen ligand aswell as on the half-life of the complex between the mutein and theligand in vivo. Further, the optimal dosage will depend on thebiodistribution of the mutein or its fusion protein or its conjugate,the mode of administration, the severity of the disease/disorder beingtreated as well as the medical condition of the patient. For example,when used in an ointment for topical applications, a high concentrationof the tear lipocalin mutein can be used. However, if wanted, the muteinmay also be given in a sustained release formulation, for exampleliposomal dispersions or hydrogel-based polymer microspheres, likePolyActive™ or OctoDEX™ (cf. Bos et al., Business Briefing: Pharmatech2003: 1-6). Other sustained release formulations available are forexample PLGA based polymers (PR pharmaceuticals), PLA-PEG basedhydrogels (Medincell) and PEA based polymers (Medivas).

Accordingly, the muteins of the present invention can be formulated intocompositions using pharmaceutically acceptable ingredients as well asestablished methods of preparation (Gennaro, A. L. and Gennaro, A. R.(2000) Remington: The Science and Practice of Pharmacy, 20th Ed.,Lippincott Williams & Wilkins, Philadelphia, Pa.). To prepare thepharmaceutical compositions, pharmaceutically inert inorganic or organicexcipients can be used. To prepare e.g. pills, powders, gelatinecapsules or suppositories, for example, lactose, talc, stearic acid andits salts, fats, waxes, solid or liquid polyols, natural and hardenedoils can be used. Suitable excipients for the production of solutions,suspensions, emulsions, aerosol mixtures or powders for reconstitutioninto solutions or aerosol mixtures prior to use include water, alcohols,glycerol, polyols, and suitable mixtures thereof as well as vegetableoils.

The pharmaceutical composition may also contain additives, such as, forexample, fillers, binders, wetting agents, glidants, stabilizers,preservatives, emulsifiers, and furthermore solvents or solubilizers oragents for achieving a depot effect. The latter is that fusion proteinsmay be incorporated into slow or sustained release or targeted deliverysystems, such as liposomes and microcapsules.

The formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile medium justprior to use.

Another aspect of the present invention relates to a method of treatinga disease or disorder, comprising administering a pharmaceuticalcomposition comprising a mutein as defined above to a subject in needthereof.

The subject in need of such a treatment may be a mammal, such as ahuman, a dog, a mouse, a rat, a pig, an ape such as cymologous to nameonly a few illustrative examples.

The precise nature of the diseases and disorders that are to be treatedaccording to the method of the invention depends on the ligand that theutilized mutein is intended to bind. Accordingly, the muteins of thepresent invention can be use to treat any disease as long as a targetmolecule that is known to be involved in the development of the diseaseor disorder can be displayed to the expression product of a nucleic acidlibrary of the present invention or displayed to otherwise obtainedmuteins of tear lipocalin.

The above described muteins binding IL-4 receptor alpha with highaffinity or pharmaceutical compositions containing them may be utilizedin a method of treating a disease or disorder associated with anincrease of the Th2 immune response. Such disease or disorder may, forexample, be an allergic reaction or an allergic inflammation. Theallergic inflammation, in turn, may be associated with allergic asthma,rhinitis, conjunctivitis or dermatitis (cf., Hage et al., CrystalStructure of the Interleukin-4 Receptor alpha chain complex reveals amosaic binding interface, Cell, Vol. 97, 271-281, Apr. 16, 1999 orMueller et al, Structure, binding and antagonists in the IL-4/IL-13receptor system, Biochemica et Biophysica Acta (2002), 237-250).

In this context it is noted that a variety of tumor cells express agreater number of high affinity IL-4 receptors than normal cells. Suchcells include solid human tumor such as melanoma, breast cancer, ovariancarcinoma, mesothelioma, glioblastoma, astrocytoma, renal cellcarcinoma, head and neck carcinoma, AIDS associated Kaposi'ssarcoma=AIDS KS, hormone dependent and independent prostate carcinomacells, and primary cultures from prostate tumors, for example (cf.,Garland L, Gitlitz B, et al., Journal of Immunotherapy. 28: 376-381, No.4, July-August 2005; Rand R W, Kreitman R J, et al. Clinical CancerResearch. 6: 2157-2165, June 2000; Husain S R, Kreitman R J, et al.Nature Medicine. 5: 817-822, July 1999; Puri R K, Hoon D S, et al.Cancer Research. 56: 5631-5637, 15 Dec. 1996, 10. Debinski W, Puri R, etal, or Husain S R, Behari N, et al. Cancer Research. 58: 3649-3653, 15Aug. 1998, Kawakami K, Leland P, et al. Cancer Research. 60: 2981-2987,1 Jun. 2000; or Strome S E, Kawakami K, et al. Clinical Cancer Research.8: 281-286, January 2002, for example. Specific examples of cells withdocuments overexpression of IL-4 receptors include, but are not limitedto, Burkitt lymphoma cell line Jijoye (B-cell lymphom), prostatecarcinoma (LNCaP, DU145), head and neck carcinoma (SCC, KCCT873),Pranceatic cancer (PANC-1 cell line), SCC-25: 13.000 (+/−500) h head andneck cancer cell line (ATCC). IL4R alpha chain plays a major role inIL4-internalization. Accordingly, when fused or conjugated to a toxin,the tear lipocalin muteins binding to IL-4 Receptor alpha chain cantherefore also be used for the treatment of tumors (cancer). Examples ofsuitable toxins include Pseudomonas exotoxin, pertussis-toxin,diphtheria toxin, ricin, saporin, pseudomonas exotoxin, calicheamicin ora derivative thereof, a taxoid, a maytansinoid, a tubulysin and adolastatin analogue. Examples of dolastatin analogues include, but arenot limited to, auristatin E, monomethylauristatin E, auristatin PYE andauristatin PHE.

For the treatment of cancer, it is also possible to conjugate muteinsbinding to IL-4 Receptor alpha chain to a cystostatic agent. Examples ofsuch cystostatic agents include Cisplatin, Carboplatin, Oxaliplatin,5-Fluorouracil, Taxotere (Docetaxel), Paclitaxel, Anthracycline(Doxorubicin), Methotrexate, Vinblastin, Vincristine, Vindesine,Vinorelbine, Dacarbazine, Cyclophosphamide, Etoposide, Adriamycine,Camptotecine, Combretatastin A-4 related compounds, sulfonamides,oxadiazolines, benzo[b]thiophenessynthetic spiroketal pyrans,monotetrahydrofuran compounds, curacin and curacin derivatives,methoxyestradiol derivatives and Leucovorin.

In this connection it is also pointed out that fusions or conjugates oftear lipocalin muteins of the invention with toxins or cystostatic agentare of course not limited to muteins with affinity to IL-4 Receptoralpha chain. Rather, as immediately evident for the person skilled inthe art, any tear lipocalin mutein that binds to a receptor expressed ona surface of cancer cells can be used in form of a fusion protein or aconjugate for the treatment of cancer.

The human tear lipocalin muteins binding VEGF-R2 or VEGF with highaffinity or pharmaceutical compositions containing them may be utilizedin a method for the treatment of a disease or disorder connected to anincreased vascularization such as cancer, neovascular wet age-relatedmacular degeneration (AMD), diabetic retinopathy or macular edema,retinopathy of prematurity or retinal vein occlusion. Such a cancer maybe selected from the group consisting of carcinomas of thegastrointestinal tract, rectum, colon, prostate, ovaries, pancreas,breast, bladder, kidney, endometrium, and lung, leukaemia, and melanoma,to name only a few illustrative examples.

As is evident from the above disclosure, a mutein of the presentinvention or a fusion protein or a conjugate thereof can be employed inmany applications. In general, such a mutein can be used in allapplications antibodies are used, except those with specifically rely onthe glycosylation of the Fc part.

Therefore, in another aspect of the invention, the invented muteins ofhuman tear lipocalin are used for the detection of a given non-naturalligand of human tear lipocalin. Such use may comprise the steps ofcontacting the mutein with a sample suspected of containing the givenligand under suitable conditions, thereby allowing formation of acomplex between the mutein and the given ligand, and detecting thecomplexed mutein by a suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins of human tear lipocalin disclosed herein may also be usedfor the separation of a given non-natural ligand of human tearlipocalin. Such use may comprise the steps of contacting the mutein witha sample supposed to contain said ligand under suitable conditions,thereby allowing formation of a complex between the mutein and the givenligand, and separating the mutein/ligand complex from the sample.

In both the use of the mutein for the detection of a given non-naturalligand as well as the separation of a given ligand, the mutein and/orthe target may be immobilized on a suitable solid phase.

The human tear lipocalin muteins of the invention may also be used totarget a compound to a preselected site. For such a purpose the muteinis contacted with the compound of interest in order to allow complexformation. Then the complex comprising the mutein and the compound ofinterest are delivered to the preselected site. This use is inparticular suitable, but not restricted to, for delivering a drug(selectively) to a preselected site in an organism, such as an infectedbody part, tissue or organ which is supposed to be treated with thedrug. Besides formation of a complex between mutein and compound ofinterest, the mutein can also be reacted with the given compound toyield a conjugate of mutein and compound. Similar to the above complex,such a conjugate may be suitable to deliver the compound to thepreselected target site. Such a conjugate of mutein and compound mayalso include a linker that covalently links mutein and compound to eachother. Optionally, such a linker is stable in the bloodstream but iscleavable in a cellular environment.

The muteins disclosed herein and its derivatives can thus be used inmany fields similar to antibodies or fragments thereof. In addition totheir use for binding to a support, allowing the target of a givenmutein or a conjugate or a fusion protein of this target to beimmobilized or separated, the muteins can be used for labeling with anenzyme, an antibody, a radioactive substance or any other group havingbiochemical activity or defined binding characteristics. By doing so,their respective targets or conjugates or fusion proteins thereof can bedetected or brought in contact with them. For example, muteins of theinvention can serve to detect chemical structures by means ofestablished analytical methods (e.g. ELISA or Western Blot) or bymicroscopy or immunosensorics. Here, the detection signal can either begenerated directly by use of a suitable mutein conjugate or fusionprotein or indirectly by immunochemical detection of the bound muteinvia an antibody.

Numerous possible applications for the inventive muteins also exist inmedicine. In addition to their use in diagnostics and drug delivery, amutant polypeptide of the invention, which binds, for example, tissue-or tumor-specific cellular surface molecules can be generated. Such amutein may, for example, be employed in conjugated form or as a fusionprotein for “tumor imaging” or directly for cancer therapy.

Thus, the present invention also involves the use of the human tearlipocalin muteins of the invention for complex formation with a givennon-natural ligand.

Another related and preferred use of a mutein described herein is targetvalidation, i.e. the analysis whether a polypeptide assumed to beinvolved in the development or progress of a disease or disorder isindeed somehow causative of that disease or disorder. This use forvalidating a protein as a pharmacological drug target takes advantage ofthe ability of a mutein of the present invention to specificallyrecognize a surface area of a protein in its native conformation, i.e.to bind to a native epitope. In this respect, it is to be noted thatthis ability has been reported only for a limited number of recombinantantibodies. However, the use of an inventive mutein for validation of adrug target is not limited to the detection of proteins as targets, butalso includes the detection of protein domains, peptides, nucleic acidmolecules, organic molecules or metal complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated by the following non-limitingExamples and the attached drawings in which:

FIG. 1 shows a map of the expression vector pTLPC10 (SEQ ID NO:1).

FIG. 2 shows the polypeptide sequence of 5148.3 J14 (SEQ ID NO:2), amutein of human tear lipocalin possessing binding affinity for the IL-4receptor alpha.

FIG. 3 shows the method of affinity screening via ELISA and the resultsobtained for muteins with affinity for IL-4 receptor alpha.

FIG. 4 shows the polypeptide sequences of the muteins with the highestaffinity for IL-4 receptor alpha (SEQ ID NOs.:3-8).

FIG. 5 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S148.3 J14; SEQ ID NO:2) to IL-4receptor alpha.

FIG. 6 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S191.5 K12; SEQ ID NO:3) to IL-4receptor alpha.

FIG. 7 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S148.3 J14AM2C2; SEQ ID NO:4) to IL-4receptor alpha.

FIG. 8 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S191.4 B24; SEQ ID NO:5) to IL-4receptor alpha.

FIG. 9 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S191.4 K19; SEQ ID NO:6) to IL-4receptor alpha.

FIG. 10 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S191.5 H16; SEQ ID NO:7) to IL-4receptor alpha.

FIG. 11 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (S197.8 D22; SEQ ID NO:8) to IL-4receptor alpha.

FIG. 12 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S148.3 J14; SEQ ID NO:2) to IL-4receptor alpha.

FIG. 13 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S191.5 K12; SEQ ID NO:3) to IL-4receptor alpha.

FIG. 14 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S148.3 J14AM2C2; SEQ ID NO:4) toIL-4 receptor alpha.

FIG. 15 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S191.4 B24; SEQ ID NO:5) to IL-4receptor alpha.

FIG. 16 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S191.4 K19; SEQ ID NO:6) to IL-4receptor alpha.

FIG. 17 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S191.5 H16; SEQ ID NO:7) to IL-4receptor alpha.

FIG. 18 shows competition ELISA measurements of the binding of a humantear lipocalin mutein of the invention (S197.8 D22; SEQ ID NO:8) to IL-4receptor alpha

FIGS. 19A to 19D show a TF-1 cell proliferation assay in presence ofIL-4 or IL-13 and human tear lipocalin muteins of the invention (S191.5K12, S148.3 J14AM2C2, S191.4 B24, S191.4 K19, S191.5 H16, and S197.8 D22[SEQ ID Nos: 3-8])

FIG. 20 shows a map of the expression vector pTLPC27 (SEQ ID NO:9).

FIGS. 21A to 21D show a proliferation assay with endothelial cellscultured from human umbilical vein (HUVEC) in presence of human VEGF165and human tear lipocalin muteins of the invention (S209.2 C23, 5209.2D16, 5209.2 N9, S209.6 H7, S209.6 H10, 5209.2 M17, S209.2 O10 [SEQ IDNOs:27-33]), wildtype tear lipocalin (gene product of pTLPC10; control)or Avastin® (Roche; control).

FIG. 22 shows BIAcore measurements of the binding of a PEGylated humantear lipocalin mutein of the invention (S148.3 J14; SEQ ID NO:2) to IL-4receptor alpha.

FIG. 23 shows BIAcore measurements of the binding of a human tearlipocalin mutein of the invention (5236.1-A22, SEQ ID NO:44) toimmobilized VEGF₈₋₁₀₉.

FIG. 24 shows BIAcore measurements of the binding of hVEGF₈₋₁₀₉,hVEGF₁₂₁, splice form hVEGF₁₆₅, and the respective mouse orthologmVEGF₁₆₄ to the human tear lipocalin mutein 5236.1-A22 (SEQ ID NO:44).

FIGS. 25A and 25B show the results of stability test of the tearlipocalin mutein S236.1-A22 (SEQ ID NO:44) in human plasma and vitreousliquid (FIG. 25A) and results of stability tests of a fusion protein ofthe mutein S236.1-A22 with an albumin-binding domain (ABD) (SEQ IDNO:51) (FIG. 25B).

FIG. 26 shows the expression vector pTLPC51 which encodes a fusionprotein comprising the OmpA signal sequence (OmpA), a mutated human tearlipocalin (Tlc), fused to an albumin-binding domain (abd), followed by aStrep-tag II.

FIG. 27 shows BIAcore measurements of the binding of tear lipocalinmutein S236.1-A22 (SEQ ID NO:44) and a fusion protein of muteinS236.1-A22 with ABD (SEQ ID NO:51) to recombinant VEGF.

FIG. 28 shows the inhibition of VEGF induced HUVEC proliferation byS236.1-A22 with ABD (SEQ ID NO:51) in the absence or presence of humanserum albumin (HSA).

FIG. 29 shows the inhibition of VEGF induced proliferation ofendothelial cells cultured from human umbilical vein (HUVEC) by thelipocalin mutein S236.1-A22 (SEQ ID NO:44) compared to the inhibitionachieved by Avastin® and wildtype tear lipocalin.

FIG. 30 shows the inhibition of VEGF mediated MAP kinase activation inHUVEC by the lipocalin mutein S236.1-A22 (SEQ ID NO:44) compared to theinhibition achieved by Avastin®.

FIG. 31 shows the results of a vascular permeability assay with localadministration of the tear lipocalin mutein S209.2_O10 (SEQ ID NO:33)compared to Avastin® and wildtype tear lipocalin.

FIG. 32 shows the results of a CAM assay comparing the median angionicindex for the tear lipocalin mutein 5209.2_O10 (SEQ ID NO:33) andAvastin® and wild type tear lipocalin.

FIG. 33 shows the concentration of lipocalin mutein in plasma in NMRImice for the tear lipocalin mutein 5236.1-A22 (SEQ ID NO:44) and afusion protein of mutein 5236.1-A22 with ABD (SEQ ID NO:51).

FIG. 34 shows the results of a vascular permeability assay aftersystemic administration of a fusion protein of tear lipocalin muteinS236.1-A22 with ABD (SEQ ID NO:51) compared to wildtype tear lipocalin,PBS buffer and Avastin®.

FIG. 35 shows the results of a tumor xenograft model (Swiss nude mice)for intraperitoneal administration of a fusion protein of tear lipocalinmutein S236.1-A22 with ABD (SEQ ID NO:51) compared to wildtype tearlipocalin, PBS buffer and Avastin®.

FIG. 36 shows the results of an Eotaxin-3 secretion assay with A549cells stimulated with IL-4 or IL-13 in the absence and presence ofincreasing concentrations of the IL-4 receptor alpha binding mutein5191.4 B24 (SEQ ID NO:4).

FIG. 37 shows the IL-4/IL-13 induced CD23 expression on stimulatedperipheral blood mononuclear cells (PBMCs) in the absence and presenceof increasing concentrations of the IL-4 receptor alpha binding muteinS191.4 B24 (SEQ ID NO:4).

FIGS. 38A and 38B show the results of a Schild analysis of the IL-4receptor alpha binding mutein S191.4 B24 (SEQ ID NO:4).

FIGS. 39A-39C show the result of an affinity assessment of the IL-4receptor alpha binding mutein S191.4 B24 (SEQ ID NO:4) for human primaryB cells.

FIG. 40 shows the results of a bioavailability test of the the IL-4receptor alpha binding mutein S191.4 B24 after intravenous, subcutaneousor intratracheal administration.

FIG. 41 shows an in vitro potency assessment of the mutein S236.1-A22(SEQ ID NO:44) with and without PEGylation with PEG20, PEG30 or PEG40 ina VEGF-stimulated HUVEC proliferation assay.

FIG. 1 shows the expression vector pTLPC10 which encodes a fusionprotein comprising the OmpA signal sequence (OmpA), the T7 affinity tagand a mutated human tear lipocalin (Tlc) followed by the Strep-tag II.Both the BstXI-restriction sites used for the cloning of the mutatedgene cassette and the restriction sites flanking the structural gene arelabeled. Gene expression is under the control of the tetracyclinepromoter/operator)(tet^(p/o)). Transcription is terminated at thelipoprotein transcription terminator (t_(lpp)). The vector furthercomprises an origin of replication (ori), the intergenic region of thefilamentous phage fl (fl-IG), the ampicillin resistance gene (amp) andthe tetracycline repressor gene (tetR). A relevant segment of thenucleic acid sequence of pTLPC10 is reproduced together with the encodedamino acid sequence in the sequence listing as SEQ ID NO:1. The segmentbegins with the XbaI restriction site and ends with the HindIIIrestriction site. The vector elements outside this region are identicalwith the vector pASK75, the complete nucleotide sequence of which isgiven in the German patent publication DE 44 17 598 A1.

FIG. 2 shows the primary structure of a human tear lipocalin mutein ofthe invention (S148.3 J14) that exhibits binding affinity for IL-4receptor alpha. The first 21 residues (underlined) constitute the signalsequence, which is cleaved upon periplasmic expression. The N-terminalT7-tag (italic) and the C-terminal Streptag-II (bold) are part of thecharacterized protein. FIG. 2 also shows that 4 N-terminal amino acidresidues (H1 H2 L3 A4) as well as the two last C-terminal amino acidresidues (S157 and D158) are deleted in this illustrative mutein of theinvention.

FIG. 3 shows results from affinity screening experiments. Monoclonalanti-StrepTag antibody (Qiagen) was coated onto the ELISA plate in orderto capture the expressed muteins of human tear lipocalin and binding ofIL-4 receptor alpha-Fc (R&D Systems; 3 nM and 0.75 nM) to the capturedmuteins was detected using an horseradish peroxidase (HRP)-conjugatedpoly clonal antibody against the Fc domain of IL-4 receptor alpha-Fc.Affinity improved clones give higher signals (left). IL-4 was coatedonto the ELISA plate and IL-4 receptor alpha-Fc (3 nM) was incubatedwith the expressed muteins. Binding of IL-4 receptor alpha-Fc having anunoccupied IL-4 binding site was detected using a HRP-conjugatedpolyclonal antibody against the Fc domain of IL-4 receptor alpha-Fc.Antagonistic affinity improved clones give lower signals (right). Thesignals corresponding to the mutein of the invention 5148.3 J14 (SEQ IDNO: 2) are marked with arrows and the signals from individual clones aredepicted by diamonds.

FIG. 4 shows the polypeptide sequences of the six muteins of human tearlipocalin with the highest binding affinity for IL-4 receptor alpha(S191.5 K12, S148.3 J14AM2C2, S191.4 B24, S191.4 K19, S191.5 H16, andS197.8 D22 [SEQ ID Nos: 3-8]) obtained by affinity maturation of SEQ IDNO:2 (S148.3 J14). The first 21 residues (underlined) of the representedprimary structure constitute the signal sequence, which is cleaved uponperiplasmic expression. The C-terminal StrepTag-II (bold) is part of thecharacterized protein. Also FIG. 4 shows that, for example, the first 4N-terminal amino acid residues (HHLA) as well as the two last C-terminalamino acid residues (SD) can be deleted in a tear lipocalin mutein ofthe invention without affecting the biological function of the protein.

FIG. 5-11 show Biacore measurements of the muteins of human tearlipocalin with affinity for IL-4 receptor alpha (S148.3 J14, S191.5 K12,S148.3 J14AM2C2, S191.4 B24, S191.4 K19, S191.5 H16, and 5197.8 D22 [SEQID Nos: 2-8]). ˜400 RU of IL-4 receptor alpha-Fc was captured on a CM-5chip, which had previously been coated with an anti human-Fc monoclonalantibody. Subsequently, mutein in different concentrations (FIG. 5: 20nM; 40 nM; 80 nM; 160 nM; 320 nM) or in a single concentration of 25 nM(FIG. 6-11) was passed over the flowcell and changes in resonance unitsrecorded. Reference signals from a flow cell that was equally treatedapart from not having any IL-4 receptor alpha-Fc was subtracted and theresulting data fitted to a 1:1 Langmuir model using the BIAevaluationsoftware. Due to the slow dissociation kinetics of the interaction inthe experiments illustrated in FIGS. 6-11 double referencing was used bysubtracting the signals from a flow cell that was equally treated apartfrom not having any IL-4 receptor alpha-Fc and subtracting the signalfrom an experiment where only sample buffer was injected. The resultingdata was fitted to a 1:1 Langmuir model with mass-transport limitationusing the BIAevaluation software. In FIGS. 6-11 the result of onerepresentative out of five experiments is shown.

FIG. 12 shows competition ELISA measurements of a human tear lipocalinmutein with binding affinity for IL-4 receptor alpha (S148.3 J14; SEQ IDNO:2). IL-4 (20 μg/ml) was coated onto an ELISA plate and IL-4 receptoralpha-Fc (15 nM) was incubated together with various concentrations ofhuman tear lipocalin mutein or IL-4 receptor-specific monoclonalantibody (MAB230, R&D Systems) for 1 h at room temperature. The IL-4receptor alpha-Fc and mutein mixture was the given to the IL-4 coatedplates for 30 min at ambient temperature. Bound IL-4 receptor alpha-Fcwas detected with a goat anti-human-Fc-HRP-conjugated antibody. The datawas fitted to the expression: 0.5*(−m0+m2−m1+sqrt((−m0+m2−m1)̂2+4*m1*m2)). Ki is given by the variable ml. The result of onerepresentative out of three experiments is shown.

FIG. 13-18 show competition ELISA measurements of the human tearlipocalin muteins with binding affinity for IL-4 receptor alpha andwildtype tear lipocalin (TLPC10; gene product of pTLPC10) as control.IL-4 receptor alpha-specific monoclonal antibody MAB230 (R&D Systems)against IL-4 receptor was coated onto an ELISA plate and biotinylatedIL-4 receptor alpha (IL-4R alpha-bio; 0.5 nM) was incubated togetherwith various concentrations of the invented muteins or TLPC10 for 1 h atambient temperature. The IL-4R alpha-bio and mutein mixture wasincubated in the MAB230-coated plates for 30 min at ambient temperature.Bound IL-4R alpha-bio was detected with Extravidin-HRP. The data werefitted to the expression: 0.5*(−m0+m2−m1+sqrt((−m0+m2−m1)̂ 2+4*m1*m2)).K_(D) is given by the variable m1. The result of one representative outof three experiments is shown.

FIG. 19 shows the results of TF-1 cell proliferation assays. TF-1 cellswere incubated for 1 hour at 37° C. with the indicated muteins, an IL-4receptor alpha-specific monoclonal antibody or a IgG2a antibody isotypecontrol in a dilution series before addition of 0.8 ng/ml IL-4 (a, b) or12 ng/ml IL-13 (c, d) for 72 h. Proliferation was measured by³H-thymidine incorporation.

FIG. 20 shows the phasmid vector pTLPC27 which encodes a fusion proteincomprising the OmpA signal sequence (OmpA), Tlc followed by theStrep-tag II, and a truncated form of the M13 coat protein pill,comprising amino acids 217 to 406 (pIII). An amber stop codon, which ispartially translated to Gln in SupE amber suppressor host strain, islocated between the Tlc coding region, including the Strep-tagII, andthe coding region for the truncated phage coat protein pill to allowsoluble expression of the Tlc mutein without the M13 coat protein pillwhen employing a non-suppressor E. coli strain. Both theBstXI-restriction sites used for the cloning of the mutated genecassette and the restriction sites flanking the structural gene arelabeled. Gene expression is under the control of the tetracyclinepromoter/operator (tet^(p/o)). Transcription is terminated at thelipoprotein transcription terminator (t_(lpp)). The vector furthercomprises an origin of replication (ori), the intergenic region of thefilamentous phage fl (fl-IG), the chloramphenicol resistance gene (cat)coding for chloramphenicol acetyl transferase and the tetracyclinerepressor gene (tetR). A relevant segment of the nucleic acid sequenceof pTLPC27 is reproduced together with the encoded amino acid sequencein the sequence listing as SEQ ID NO:9.

FIG. 21 shows the results of a proliferation assay employing the humantear lipocalin muteins with binding affinity for human VEGF, wildtypetear lipocalin (TLPC10) or VEGF-specific therapeutic antibody Avastin®.Approximately 1.400 HUVEC cells were seeded in complete medium and afterovernight incubation at 37° C., cells were washed and basal mediumcontaining 0.5% FCS, hydrocortisone and gentamycin/amphotericin wasadded. VEGF-specific mutein 5209.2-C23, 5209.2-D16, 5209.2-N9,S209.6-H7, S209.6-H10, S209.2-M17, S209.2-O10 (SEQ ID NOs:27-33),wildtype tear lipocalin (gene product of pTLPC10; as control) ortherapeutic VEGF-specific monoclonal antibody Avastin® (Roche; ascontrol) was added at the indicated concentration in triplicate wells.After 30 min, either human VEGF165 or human FGF-2, as a control forproliferation not induced by VEGF (not shown), was added and theviability of the cells was assessed after 6 days with CellTiter 96Aqueous One chromogenic assay (Promega).

FIG. 22 shows Biacore measurements of the PEGylated mutein S148.3 J14(SEQ ID NO:2) of human tear lipocalin with affinity for IL-4 receptoralpha. ˜400 RU of IL-4 receptor alpha-Fc was captured on a CM-5 chip,which had previously been coated with an anti human-Fc monoclonalantibody. Subsequently, mutein in different concentrations (200 nM; 67nM; 22 nM was passed over the flowcell and changes in resonance unitswere recorded. Reference signals from a flow cell that was equallytreated apart from not having any IL-4 receptor alpha-Fc was subtractedand the resulting data were fitted to a 1:1 Langmuir model using theBIAevaluation software.

FIG. 23 shows exemplary Biacore measurements of the binding of humantear lipocalin mutein S236.1-A22 (SEQ ID NO:44) to immobilizedVEGF₈₋₁₀₉. VEGF₈₋₁₀₉ was immobilized on a CMS chip using standard aminechemistry. Lipocalin mutein S236.1-A22 was applied with a flow rate of30 μl/min at six concentrations from 500 nM to 16 nM. Evaluation ofsensorgrams was performed with BIA T100 software to determine k_(on),k_(off) and K_(D) of the mutein.

FIG. 24 shows affinity measurements of the mutein S236.1-A22 (SEQ IDNO:44) that was immobilized on a sensor chip with different forms ofVEGF. Affinity measurements were performed essentially as described inExample 9 of WO 2006/56464 with the modifications that the mutein wasimmobilized and 70 μl of sample containing the different VEGF variantswas injected at a concentration of 250 nM. The qualitative comparison ofthe results illustrate that the truncated form hVEGF₈₋₁₀₉ and hVEGF₁₂₁show basically identical sensorgrams indicating similar affinity to thetear lipocalin mutein S236.1-A22 (SEQ ID NO:44). The splice formhVEGF₁₆₅ also shows strong binding to the lipocalin mutein, while therespective mouse ortholog mVEGF₁₆₄ has slightly reduced affinity.

FIG. 25 shows a stability test of VEGF-binding mutein S236.1-A22 at 37°C. in PBS and human serum that was performed essentially as described inExample 15 of the International patent application WO2006/056464 exceptthat the concentration utilized was 1 mg/ml. No alteration of the muteincould be detected during the seven day incubation period in PBS asjudged by HPLC-SEC (data not shown). Incubation of the lipocalin muteinin human serum resulted in a drop of affinity after 7 days to approx.70% compared to the reference (FIG. 25A). The stability of theABD-fusion of S236.1-A22 (SEQ ID NO: 51) in human serum was also testedas described above. No loss of activity could be detected during theseven day incubation period (FIG. 25B)

FIG. 26 shows the expression vector pTLPC51 which encodes a fusionprotein comprising the OmpA signal sequence (OmpA), a mutated human tearlipocalin (Tlc), fused to an albumin-binding domain (abd), followed by aStrep-tag II. Both the BstXI-restriction sites used for the cloning ofthe mutated gene cassette and the restriction sites flanking thestructural gene are labeled. Gene expression is under the control of thetetracycline promoter/operator)(tet^(p/o)). Transcription is terminatedat the lipoprotein transcription terminator (t_(lpp)). The vectorfurther comprises an origin of replication (ori), the intergenic regionof the filamentous phage fl (fl-IG), the ampicillin resistance gene(amp) and the tetracycline repressor gene (tetR). A relevant segment ofthe nucleic acid sequence of pTLPC51 is reproduced together with theencoded amino acid sequence in the sequence listing as SEQ ID NOs:48 and49. The segment begins with the XbaI restriction site and ends with theHindIII restriction site. The vector elements outside this region areidentical with the vector pASK75, the complete nucleotide sequence ofwhich is given in the German patent publication DE 44 17 598 A1.

FIG. 27 shows affinity measurements of the ABD-fusion of tear lipocalinmutein S236.1-A22 (A22-ABD) (SEQ ID NO: 51) (200 pM) towards recombinantVEGF₈₋₁₀₉ using surface plasmon resonance (Biacore). Affinitymeasurements were performed essentially as described in Example 9 of WO2006/56464 with the modifications that approximately 250 RU ofrecombinant VEGF₈₋₁₀₉ was directly coupled to the sensor chip usingstandard amine chemistry. 40 μl of the mutein was injected at aconcentration of 400 nM. The affinity was found basically unaltered andmeasured to be 260 pM.

FIG. 28 shows a test of the functionality of the lipocalin muteinA22-ABD (ABD-fusion of S236.1-A22) in the presence of human serumalbumin by asessing its ability to inhibit VEGF induced HUVECproliferation. HUVEC (Promocell) were propagated on gelatine-coateddishes and used between passages P2 and P8. On day 1, 1400 cells wereseeded per well in a 96 well plate in complete medium. On day 2, cellswere washed and 100 μl of basal medium containing 0.5% FCS,hydrocortisone and gentamycin/amphotericin was added. Proliferation wasstimulated with 20 ng/ml VEGF₁₆₅ or 10 ng/ml FGF-2 which were mixed withthe lipocalin mutein 5236.1-A22-ABD (SEQ ID NO:51), incubated for 30 minand added to the wells. Viability was determined on day 6 and theresults expressed as % inhibition. Human serum albumin (HSA, 5 μM) wasadded where indicated. At 5 μM HSA, >99.8% of A22-ABD is associated withHSA at any given time.

FIG. 29 shows the inhibition of VEGF induced HUVEC proliferation bymuteins of the invention. HUVEC (Promocell) were propagated ongelatine-coated dishes and used between passages P2 and P8. On day 1,1400 cells were seeded per well in a 96 well plate in complete medium.On day 2, cells were washed and 100 μl of basal medium containing 0.5%FCS, hydrocortisone and gentamycin/amphotericin was added. Proliferationwas stimulated with 20 ng/ml VEGF165 or 10 ng/ml FGF-2 which were mixedwith the lipocalin mutein S236.1-A22 (SEQ ID NO:44), incubated for 30min and added to the wells. Viability was determined on day 6 and theresults expressed as % inhibition.

FIG. 30 shows the Inhibition of VEGF-mediated MAP Kinase activation inHUVEC by muteins of the present invention. HUVEC were seeded in 96-wellplates at 1,400 cells per well in standard medium (Promocell,Heidelberg). On the following day, FCS was reduced to 0.5% andcultivation was continued for 16 h. Cells were then starved in 0.5% BSAin basal medium for 5 h. HUVEC were stimulated with VEGF₁₆₅ (Reliatech,Braunschweig) for 10 min in the presence of increasing concentrations oftear lipocalin mutein A22 or Avastin (bevacizumab, Genentech/Roche) inorder to obtain a dose-response curve. Phosphorylation of the MAPkinases ERK1 and ERK2 was quantified using an ELISA according to themanufacturer's manual (Active Motif, Rixensart, Belgium). The IC 50value was determined to be 4.5 nM for the mutein A22 (SEQ ID NO:44) and13 nM for Avastin®.

FIG. 31 shows a vascular permeability assay with local administration oftear lipocalin mutein. Duncan-Hartley guinea pigs weighing 350±50 g wereshaved on the shoulder and on the dorsum. The animals received anintravenous injection via the ear vein of 1 ml of 1% Evan's Blue dye.Thirty minutes later 20 ng VEGF₁₆₅ (Calbiochem) was mixed with testsubstance or control article at a tenfold molar excess and injectedintradermally on a 3×4 grid. Thirty minutes later, animals wereeuthanized by CO₂ asphyxiation. One hour after the VEGF injections, theskin containing the grid pattern was removed and cleaned of connectivetissue. The area of dye extravasation was quantified by use of an imageanalyzer (Image Pro Plus 1.3, Media Cybernetics).

FIG. 32 shows a chick chorioallantoic membrane (CAM) assay. Collagenonplants containing FGF-2 (500 ng), VEGF (150 ng) and tear lipocalinmutein (1.35 μg) or Avastin (10 μg) as indicated were placed onto theCAM of 10 day chicken embryos (4/animal, 10 animals/group). At 24 h thetear lipocalin mutein or Avastin were reapplied topically to the onplantat the same dose. After 72 h onplants were collected and images werecaptured. The percentage of positive grids containing at least onevessel was determined by a blinded observer. The median angiogenic indexis reported for the VEGF antagonists S209.2-O10 (SEQ ID NO:33) andAvastin® as well as wild type tear lipocalin control as the fraction ofpositive grids.

FIG. 33 shows the determination of pharmacokinetic (PK) parameters forA22 and A22-ABD in mice. Pharmacokinetic (PK) parameters (half-lifeplasma concentration, bioavailibity) for tear lipocalin mutein 5236.1A22 (SEQ ID NO:44) (4 mg/kg) after i.v. and the fusion protein ofmuteiin 5236.1 A22 with ABD (SEQ ID NO:51) (5.4 mg/kg) following i.v. ori.p. single bolus administration were determined in NMRI mice. Plasmawas prepared from terminal blood samples taken at pre-determinedtimepoints and the concentrations of the lipocalin mutein weredetermines by ELISA. Results were analyzed using WinNonlin software(Pharsight Corp., Mountain View, USA). T_(1/2), A22 i.v.: 0.42 h;T_(1/2) A22-ABD i.v.: 18.32 h; T_(1/2), A22-ABD i.p.: 20.82 h. Thebioavailability following i.p. administration of the fusion proteinA22-ABD was 82.5%.

FIG. 34 shows a vascular permeability assay with systemic administrationof tear lipocalin mutein. Twelve hours prior to the experiment, testsubstances or controls were injected intravenously into 3 animals pergroup. Group 1: PBS vehicle; Group 2: Avastin, 10 mg/kg; Group 3: mutein5236.1 A22-ABD, 6.1 mg/kg; Group 4: TLPC51: 6.1 mg/kg. At time=0 Evan'sBlue was injected. Thirty minutes later, 4 doses of VEGF (5, 10, 20 or40 ng) were injected intradermally in triplicate on a 3×4 grid. Thirtyminutes after the VEGF injections the animals were sacrificed and dyeextravasation was quantified by use of an image analyzer (Image Pro Plus1.3, Media Cybernetics).

FIG. 35 shows the effect of the muteins of the invention in a tumorxenograft model. Irradiated (2.5 Gy, Co⁶⁰) Swiss nude mice wereinoculated subcutaneously with 1×10⁷ A673 rhabdomyosarcoma cells (ATTC)in matrigel into the right flank (n=12 per group). Treatments wereadministered intraperitoneally and were initiated on the same day andcontinued for 21 days. Group 1: PBS vehicle, daily; Group 2: Avastin(bevacizumab, Genentech/Roche), 5 mg/kg every 3 days; Group 3: lipocalinmutein A22-ABD (SEQ ID NO:51), daily, 3.1 mg/kg; Group 4: TLPC51, daily,3.1 mg/kg. The dose of the lipocalin mutein A22-ABD was chosen toachieve the constant presence of an equimolar number of VEGF bindingsites of the mutein and Avastin based on the A22-ABD PK data andestimated serum half life of antibodies in mice. Tumor size was measuredtwice weekly with a calliper and the tumor volume was estimatedaccording to the formula (length×width²)/2. Mice were sacrificed whenthe tumor volume exceeded 2,000 mm³.

FIG. 36 shows the results of an Eotaxin-3 secretion assay with A549cells. A549 cells were stimulated with 0.7 nM IL-4 or 0.83 nM IL-13respectively in the absence and presence of increasing concentrations ofthe IL-4 receptor alpha binding mutein S191.4 B24 (SEQ ID NO:4).Eotaxin-3 secretion was assessed after 72 hours by measuring Eotaxin 3concentrations in the cell culture supernatent using a commerciallyavailable kit.

FIG. 37 shows the IL-4/IL-13 induced CD23 expression on stimulatedperipheral blood mononuclear cells (PBMCs) after 48 h in the absence andpresence of increasing concentrations of the IL-4 receptor alpha bindingmutein S191.4 B24 (SEQ ID NO:4). Total human PBMCs were isolated frombuffy coat. Increasing concentrations of the IL-4 receptor alpha bindingmutein S191.4 B24 were added and cells were stimulated with IL-4 orIL-13 at final concentrations of 1.0 nM or 2.5 nM, respectively. After48 hours, activated, CD23 expressing CD14⁺ monocytes were quantified byflow cytometry.

FIG. 38 shows the results of a Schild analysis of the IL-4 receptoralpha binding mutein S191.4 B24 (SEQ ID NO:4). IL-4 dose dependentproliferation of TF-1 cells was assessed in the absence or presence ofseveral fixed concentrations of the IL-4 receptor alpha binding muteinS191.4 B24 (FIG. 38A). The Schild analysis of the obtained results (FIG.38B) yielded a K_(d) of 192 pM (linear regression) and 116 pM(non-linear regression).

FIG. 39 shows the result of an affinity assessment of the IL-4 receptoralpha binding mutein 5191.4 B24 (SEQ ID NO:4) for human primary B cells.PBMCs were isolated from human blood and incubated with differentconcentrations of the IL-4 receptor alpha binding human tear lipocalinmutein S191.4 B24 or the wild-type human tear lipocalin (TLPC26). Cellswere then stained with anti-CD20-FITC monoclonar antibodies and abiotinylated anti-lipocalin antiserum, followed by streptavidin-PE.Results for the wild-type lipocalin and the IL-4 receptor alpha bindinglipocalin mutein S191.4 B24 are shown in FIGS. 39A and 39B,respectively. The determined percentage of PE-positive B cells wasfitted against the concentration of the lipocalins (FIG. 39C) and theEC₅₀ calculated from the obtained curve. The EC₅₀ of the IL-4 receptoralpha binding mutein S191.4 B24 (SEQ ID NO:4) was calculated as 105 pM.

FIG. 40 shows the results of a bioavailability test of the the IL-4receptor alpha binding mutein S191.4 B24 after intravenous, subcutaneousor intratracheal administration. Sprague-Dawley rats received a singledose of the mutein S191.4 B24 at 4 mg/kg via the indicated routes.Intratracheal administration was performed with a microspray dosingdevice (PennCentury, USA). Plasma samples were obtained at predeterminedtime points and subjected to a sandwich ELISA analysis in order todetermine the remaining concentrations of the functionally activemutein. Concentrations were analyzed by non-compartmental PK analysis.Bioavailability was 100% after subcutaneous administration and 13.8%following intratracheal delivery.

FIG. 41 shows an in vitro potency assessment of the mutein 5236.1-A22(SEQ ID NO:44) either unPEGylated or PEGylated with PEG20, PEG30 orPEG40 compared to human tear lipocalin wt. The IC₅₀ values weredetermined via titration of the respective human tear lipocalin muteinin a VEGF-stimulated HUVEC proliferation assay and determining theproliferation inhibition.

EXAMPLES

Unless otherwise indicated, established methods of recombinant genetechnology were used, for example, as described in Sambrook et al.(supra).

Example 1 Generation of a Library with 2×10⁹ Independent Tlc Muteins

A random library of tear lipocalin (Tlc) with high complexity wasprepared by concerted mutagenesis of the 18 selected amino acidpositions 26, 27, 28, 29, 30, 31, 32, 33, 34, 56, 57, 58, 80, 83, 104,105, 106, and 108 of the mature wild type human tear lipocalin. To thisend, a gene cassette wherein the corresponding codons were randomized ina targeted fashion was assembled via polymerase chain reaction (PCR)with degenerate primer oligodeoxynucleotides in two steps according to astrategy described before (Skerra, A. (2001) “Anticalins”: a new classof engineered-ligand-binding proteins with antibody-like properties. J.Biotechnol. 74, 257-275). In this library design the first 4 N-terminalamino acid residues (HHLA) as well as the two last C-terminal amino acidresidues (SD) of the wild type sequence of tear lipocalin were deleted(for this reason, all tear lipocalin muteins shown in the attachedSequence Listing have Ala5 of the wild type sequence as N-terminalresidue and Gly 156 as C-terminal residue (the latter optionally fusedto an affinity tag, for example)).

In the first step of the generation of the random library, a PCRfragment with randomized codons for the first and second exposed loop ofTlc was prepared using primers TL46 (SEQ ID NO:10) and TL47 (SEQ IDNO:11) while another PCR fragment with randomized codons for the thirdand fourth exposed loop of Tlc was prepared in parallel, using primersTL48 (SEQ ID NO:12) and TL49 (SEQ ID NO:13). In the second step thesetwo PCR fragments were combined with a connecting oligodeoxynucleotideand used as templates in a PCR reaction with primers AN-14 (SEQ IDNO:14), TL50 bio (SEQ ID NO:15) and TL51 bio (SEQ ID NO:16) to yield theassembled randomized gene cassette.

The two PCR reactions (1a and 1b) for the first step were each performedin a volume of 100 μl using 10 ng pTLPC10 plasmid DNA (FIG. 1) for eachreaction as template, together with 50 pmol of each pair of primers(TL46 and TL47, or TL48 and TL49, respectively), which were synthesizedaccording to the conventional phosphoramidite method. In addition, thereaction mixture contained 10 μl 10× Taq reaction buffer (100 mMTris/HCl pH 9.0, 500 mM KCl, 15 mM MgCl₂, 1% v/v Triton X-100) and 2 μldNTP-Mix (10 mM dATP, dCTP, dGTP, dTTP). After bringing to volume withwater, 5 u Taq DNA polymerase (5 u/μl, Promega) were added and 20 cyclesof 1 minute at 94° C., 1 minute at 58° C. and 1.5 minutes at 72° C. werecarried out in a programmable thermocycler with a heated lid(Eppendorf), followed by an incubation for 5 minutes at 60° C. forcompletion. The amplification products with the desired size of 135 bpand 133 bp, respectively, were isolated by preparative agarose gelelectrophoresis using GTQ Agarose (Roth) and the Wizard DNA extractionkit (Promega).

For the second PCR step a 1000 μl mixture was prepared, whereinapproximately 500 fmol of both fragments from PCR reactions 1a and 1bwere used as templates in the presence of 500 pmol of each of theflanking primers TL50 bio (SEQ ID NO:15) and TL51 bio (SEQ ID NO:16) and10 pmol of the mediating primer AN-14 (SEQ ID NO:14). Both flankingprimers carried a biotin group at their 5′-ends, thus allowing theseparation of the PCR product after BstXI cleavage from incompletelydigested product via streptavidin-coated paramagnetic beads. Inaddition, the reaction mix contained 100 μl 10× Taq buffer, 20 μldNTP-Mix (10 mM dATP, dCTP, dGTP, dTTP), 50 u Taq DNA polymerase (5u/μl, Promega) and water to bring it to the final volume of 1000 μl. Themixture was divided into 100 μl aliquots and PCR was performed with 20cycles of 1 minute at 94° C., 1 minute at 57° C., 1.5 minutes at 72° C.,followed by a final incubation for 5 minutes at 60° C. The PCR productwas purified using the E.Z.N.A. Cycle-Pure Kit (PeqLab).

For subsequent cloning, this fragment representing the central part ofthe library of Tlc muteins in nucleic acid form was first cut with therestriction enzyme BstXI (Promega) according to the instructions of themanufacturer and then purified by preparative agarose gelelectrophoresis as described above, resulting in a double-strandedDNA-fragment of 301 base pairs in size.

DNA fragments not or incompletely digested were removed via their5′-biotin tags using streptavidin-coated paramagnetic beads (Merck). Tothis end, 150 μl of the commercially available suspension of thestreptavidin-coated paramagnetic particles (at a concentration of 10mg/ml) was washed three times with 100 μl TE buffer (10 mM Tris/HCl pH8.0, 1 mM EDTA). The particles were then drained with the help of amagnet and mixed with 70 pmol of the digested DNA fragment in 100 μl TEbuffer for 15 minutes at room temperature. The paramagnetic particleswere then collected at the wall of the Eppendorf vessel with the aid ofa magnet and the supernatant containing the purified, fully digested DNAfragment was recovered for use in the following ligation reaction.

The vector pTLPC27 (FIG. 20) was cut with the restriction enzyme BstXI(Promega) according to the instructions of the manufacturer and theobtained large vector fragment was purified by preparative agarose gelelectrophoresis as described above, resulting in a double-strandedDNA-fragment of 3772 base pairs in size representing the vectorbackbone.

For the ligation reaction, 40 pmol of the PCR fragment and 40 pmol ofthe vector fragment (pTLPC27) were incubated in the presence of 1074Weiss Units of T4 DNA ligase (Promega) in a total volume of 10.76 ml (50mM Tris/HCl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 50 μg/ml BSA) for48 h at 16° C. The DNA in the ligation mixture was then precipitated 1.5h by adding 267 μl yeast tRNA (10 mg/ml solution in H₂O (Roche)), 10.76ml 5 M ammonium acetate, and 42.7 ml ethanol. After precipitation, theDNA pellet was washed with 70% EtOH and then dried. At the end the DNAwas dissolved to a final concentration of 200 μg/ml in a total volume of538 μl of water.

The preparation of electrocompetent bacterial cells of E. colistrainXL1-Blue (Bullock et al., supra) was carried out according to themethods described by Tung and Chow (Trends Genet. 11 (1995), 128-129)and by Hengen (Trends Biochem. Sci. 21 (1996), 75-76). 11 LB medium (10g/L Bacto Tryptone, 5 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5) wasadjusted to an optical density at 600 nm of OD₆₀₀=0.08 by addition of anovernight culture of XL1-Blue and was incubated at 140 rpm and 26° C. ina 21 Erlenmeyer flask. After reaching an OD₆₀₀=0.6, the culture wascooled for 30 minutes on ice and subsequently centrifuged for 15 minutesat 4000 g and 4° C. The cells were washed twice with 500 ml ice-cold 10%w/v glycerol and finally re-suspended in 2 ml of ice-cold GYT-medium(10% w/v glycerol, 0.125% w/v yeast extract, 0.25% w/v tryptone). Thecells were then aliquoted (200 up, shock-frozen in liquid nitrogen andstored at −80° C.

Electroporation was performed with a Micro Pulser system (BioRad) inconjunction with cuvettes from the same vendor (electrode distance 2 mm)at 4° C. Aliquots of 10 μl of the ligated DNA solution (containing 1 μgDNA) was mixed with 100 μl of the cell suspension, first incubated for 1minute on ice, and then transferred to the pre-chilled cuvette.Electroporation was performed using parameters of 5 ms and 12.5 kV/cmfield strength and the suspension was immediately afterwards diluted in2 ml ice-cold SOC medium (20 g/L Bacto Tryptone, 5 g/L Bacto YeastExtract, 10 mM NaCl, 2.5 mM KCl, pH 7.5, autoclaved, beforeelectroporation 10 ml/L 1 M MgCl₂ and 1 M MgSO₄ with 20 ml/L 20% Glucosewere added), followed by incubation for 60 min at 37° C. and 140 rpm.After that, the culture was diluted in 2 L 2×YT medium (16 g/L BactoTryptone, 10 g/L Bacto Yeast Extract, 5 g/L NaCl, pH 7.5) containing 100μg/ml chloramphenicol (2 YT/Cam), resulting in an OD₅₅₀ of 0.26. Theculture was incubated at 37° C. until the OD₅₅₀ had risen again by 0.6units.

By employing a total of 107.6 μg ligated DNA in 54 electroporation runs,a total of about 2.0×10⁹ transformants were obtained. The transformantswere further used for the preparation of phagemids coding for thelibrary of the Tlc muteins as fusion proteins.

For preparation of the phagemid library, 41 of the culture from abovewere infected with 1.3×10¹² pfu VCS-M13 helper phage (Stratagene). Afteragitation at 37° C. for 45 min the incubation temperature was lowered to26° C. After 10 min of temperature equilibration 25 μg/1anhydrotetracycline was added in order to induce gene expression for thefusion protein between the Tlc muteins and the phage coat protein.Phagemid production was allowed for 11 h at 26° C. After removal of thebacteria by centrifugation the phagemids were precipitated from theculture supernatant twice with 20% (w/v) polyethylene glycol 8000(Fluka), 15% (w/v) NaCl and finally dissolved in PBS (4 mM KH₂PO₄, 16 mMNa₂HPO₄, 115 mM NaCl).

Example 2 Phagemid Presentation and Selection of Tlc Muteins withAffinity for IL-4 Receptor Alpha

Phagemid display and selection was performed employing the phagemidsobtained from Example 1 essentially as described in WO 2006/56464Example 2 with the following modifications: The target protein (IL-4receptor alpha, Peprotech) was employed at a concentration of 200 nM andwas presented to the library as biotinylated protein with subsequentcapture of the phage-target complex using streptavidin beads (Dynal).Alternatively, the target protein was employed as Fc-fusion protein(IL-4 receptor alpha-Fc, R&D Systems) at a concentration of 200 nM andsubsequent capture of the phage-target complex using protein G beads(Dynal) and by immobilization of Fc-fusion protein on anti-human Fccapture antibody (Jackson Immuno Research) coated immunosticks (Nunc)according to the instructions of the manufacturer. Three or four roundsof selection were performed.

Example 3 Identification of IL-4 Receptor Alpha-Specific Muteins UsingHigh-Throughput ELISA Screening

Screening of the muteins selected according to Example 2 was performedessentially as described in Example 3 of WO 2006/56464 with thefollowing modifications: Expression vector was pTLPC10 (FIG. 1). Targetprotein used was IL-4 receptor alpha-Fc (R&D Systems) and IL-4 receptoralpha (Peprotech) both at 2 μg/ml.

Screening 5632 clones, selected as described in Example 2, lead to theidentification of 2294 primary hits indicating that successful isolationof muteins from the library had taken place. Using this approach theclone S148.3 J14 (SEQ ID NO:2) was identified. The sequence of S148.3J14 is also depicted in FIG. 2.

Example 4 Affinity Maturation of the Mutein 5148.3 J14 Using Error-PronePCR

Generation of a library of variants based on the mutein S148.3 J14 (SEQID NO:2) was performed essentially as described in Example 5 of WO2006/56464 using the oligonucleotides TL50 bio (SEQ ID NO:15) and TL51bio (SEQ ID NO:16) resulting in a library with 3 substitutions perstructural gene on average.

Phagemid selection was carried out as described in Example 2 butemploying limited target concentration (2 nM, 0.5 nM and 0.1 nM of IL-4receptor alpha, Peprotech Ltd), extended washing times together with anantagonistic monoclonal antibody against IL-4 receptor alpha (MAB230,R&D Systems; 1 hour washing time and 2 hours washing time) or shortincubation times (30 seconds, 1 minute and 5 minutes). Three or fourrounds of selection were performed.

Example 5 Affinity Maturation of the Mutein 5148.3 J14 Using aSite-Directed Random Approach

A library of variants based on the mutein 5148.3 J14 (SEQ ID NO:2) wasdesigned by randomization of the positions 34, 53, 55, 58, 61, 64 and 66to allow for all 20 amino acids on these positions. The library wasconstructed essentially as described in Example 1 with the modificationthat the deoxynucleotides TL70 (SEQ ID NO:17), TL71 (SEQ ID NO:18) andTL72 (SEQ ID NO:19) were used instead of TL46, TL47, and AN-14,respectively.

Phagemid selection was carried out as described in Example 2 usinglimited target concentration (0.5 nM and 0.1 nM of IL-4 receptor alpha,Peprotech) combined with extended washing times together with acompetitive monoclonal antibody against IL-4 receptor alpha (MAB230, R&DSystems; 1 hour washing) or short incubation times (10 minutes),respectively. Three or four rounds of selection were performed.

Example 6 Affinity Screening of IL-4 Receptor Alpha-Binding MuteinsUsing High-Throughput ELISA Screening

Screening was performed as described in Example 3 with the modificationthat a concentration of 3 nM IL-4 receptor alpha-Fc (R&D Systems) wasused and the additions that i) a monoclonal anti-Strep tag antibody(Qiagen) was coated onto the ELISA plate in order to capture theproduced muteins and binding of IL-4 receptor alpha-Fc (R&D Systems, 3nM and 0.75 nM) to the captured muteins of tear lipocalin was detectedusing a HRP (horseradish peroxidase)-conjugated poly clonal antibodyagainst the Fc domain of IL-4 receptor alpha-Fc. Additionally in analternative screening setup ii) IL-4 was coated onto the ELISA plate andIL-4 receptor alpha-Fc (R&D Systems, 3 nM) was incubated with theexpressed muteins and binding of IL-4 receptor alpha-Fc with anunoccupied IL-4 binding site was detected using a HRP-conjugatedpolyclonal antibody against the Fc domain of IL-4 receptor alpha-Fc.

A result from such a screen is depicted in FIG. 3. A large number ofmuteins selected as described in Example 4 and 5 were identified havingimproved affinity for IL-4 receptor alpha as compared to the muteinS148.3 J14 (SEQ ID NO:2) which served as the basis for affinitymaturation. Using this approach the muteins S191.5 K12, S191.4 B24,S191.4 K19, S191.5 H16, S197.8 D22 and S148.3 J14AM2C2 (SEQ ID NOs.:3-8)were identified. The sequences of 5191.5 K12, 5191.4 B24, 5191.4 K19,5191.5 H16, 5197.8 D22 and 5148.3 J14AM2C2 are also depicted in FIG. 4.

Example 7 Production of IL-4 Receptor Alpha-Binding Muteins

For preparative production of IL-4 receptor alpha-specific muteins, E.coli K12 strain JM83 harbouring the respective mutein encoded on theexpression vector pTLPC10 (FIG. 1) was grown in a 2 L shake flaskculture in LB-Ampicillin medium according to the protocol described inSchlehuber, S. et al. (J. Mol. Biol. (2000), 297, 1105-1120). Whenlarger amounts of protein were needed, the E. coli strain W3110harbouring the respective expression vector was used for theperiplasmatic production via bench top fermenter cultivation in a 11 or101 vessel based on the protocol described in Schiweck, W., and Skerra,A. Proteins (1995) 23, 561-565).

The muteins were purified from the periplasmic fraction in a single stepvia streptavidin affinity chromatography using a column of appropriatebed volume according to the procedure described by Skerra, A. & Schmidt,T. G. M. (2000) (Use of the Strep-tag and streptavidin for detection andpurification of recombinant proteins. Methods Enzymol. 326A, 271-304).To achieve higher purity and to remove any aggregated recombinantprotein, a gel filtration the muteins was finally carried out on aSuperdex 75 HR 10/30 column (24-m1 bed volume, Amersham PharmaciaBiotech) in the presence of PBS buffer. The monomeric protein fractionswere pooled, checked for purity by SDS-PAGE, and used for furtherbiochemical characterization.

Example 8 Affinity Measurement Using Biacore

Affinity measurements were performed essentially as described in Example9 of WO 2006/56464 with the modifications that approximately 400 RU ofIL-4 receptor alpha-Fc (R&D Systems) was immobilized (instead of 2000 RUof human CTLA-4 or murine CTLA-4-Fc used as target in WO 2006/56464) and100 μl of mutein was injected at a concentration of 25 nM (instead of 40μl sample purified lipocalin muteins at concentrations of 5-0.3 μM asused in WO 2006/56464).

Results from the affinity measurements employing S148.3 J14, S191.5 K12,S191.4 B24, S191.4 K19, S191.5 H16, S197.8 D22 and S148.3 J14AM2C2 aredepicted in FIGS. 5-11 and are summarized in Table I.

TABLE I Affinities of selected muteins of the invention for IL-4receptor alpha as determined by Biacore. Averages (standard deviation)of five experiments are shown. Affinity Biacore k_(on) k_(off) Clone(pM) (1/Ms × 10⁵) (1/s × 10⁻⁵) S148.3 J14 37500 1.4 517 S191.5 K12 13.5(2.9) 58 (27)  7.7 (3.3) S148.3 AM2C2 17.9 (2.7) 23 (1.7) 4.2 (0.7)S191.4 B24 19.3 (3.3) 26 (6.7) 4.9 (1.0) S191.4 K19 20.1 (14)  17 (2.7)3.6 (2.8) S191.5 H16 24.3 (12)  17 (1.8) 4.1 (1.6) S197.8 D22 55.8 (4.2)11 (1.3) 6.3 (1.0)

Example 9 Identification of Antagonists of IL-4 Using an InhibitionELISA

Inhibition of the interaction between IL-4 and IL-4 receptor alpha bythe selected muteins was evaluated in an inhibition ELISA. Therefore, aconstant concentration of IL-4 receptor alpha (0.5 nM biotinylated IL-4receptor alpha, Peprotech, or 15 nM IL-4 receptor alpha-Fc, R&D Systems)was incubated with a dilution series of tear lipocalin mutein and theamount of IL-4 receptor alpha with an unoccupied IL-4 binding site wasquantified in an ELISA where the plate had been coated with IL-4 or anantagonistic anti IL-4 receptor alpha monoclonal antibody. Boundbiotinylated IL-4 receptor alpha was detected using HRP-conjugatedExtravidin (Sigma) and compared to a standard curve of defined amountsof biotinylated IL-4 receptor alpha. Results from measurements employingthe muteins of S148.3 J14, S191.5 K12, S191.4 B24, S191.4 K19, S191.5H16, S197.8 D22 and S148.3 J14AM2C2 are depicted in FIGS. 12-18 and aresummarized in Table II.

TABLE II Antagonistic ability and affinities for IL-4 receptor alpha ofselected tear lipocalin muteins of the invention as determined bycompetition ELISA. Averages (standard deviation) of three experimentsare shown. Clone Affinity Competition ELISA (pM) S148.3 J14 17300 S191.5K12 25.3 (9.9) S148.3 AM2C2 40.7 (14.8) S191.4 B24 49.2 (14) S191.4 K19120 (32) S191.5 H16 61.7 (11.4) S197.8 D22 140 (37)

Example 10 Identification of Antagonists of IL-4 and IL-13 SignallingUsing a TF-1 Proliferation Assay

IL-4 and IL-13-stimulated TF-1 cell proliferation assays were performedessentially as described in Lefort et al. (Lefort S., Vita N., Reeb R.,Caput D., Ferrara P. (1995) FEBS Lett. 366(2-3), 122-126). The resultsfrom a TF-1 proliferation assay is depicted in FIG. 19 and shows thatthe high affinity variants S191.5 K12, S191.4 B24, S191.4 K19, S191.5H16, S197.8 D22 and 5148.3 J14AM2C2 are potent antagonists of IL-4 aswell as IL-13 induced signalling and proliferation.

Example 11 Anti-IL-4 Receptor Alpha Muteins of Human Tear LipocalinInhibit the STAT6 Mediated Pathway

TF-1 cells were cultured in RPMI 1640 containing 10% heat-inactivatedfetal calf serum, 2 mM L-glutamine, 100 Units/ml penicillin, 100 μg/mlstreptomycin and supplemented with 2 ng/ml recombinant humangranulocyte-macrophage colony-stimulating factor. The cells were seededat 5×10⁴ cells/ml in a total volume of 20 ml medium in 100 mm diametertissue culture dishes, split and reseeded at this concentration every 2to 3 days and cultured at 37° C. in a humidified atmosphere of 5% CO₂.

TF-1 cells were harvested by centrifugation at 1200 rpm for 5 min andwashed twice by centrifugation at 1200 rpm for 5 min in RPMI 1640containing 1% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100Units/ml penicillin and 100 μg/ml streptomycin (RPMI-1% FCS). Cells wereresuspended at 1×10⁶ cells/ml in RPMI-1% FCS, plated out at 1 ml in 24well plates and cultured overnight. On the following day, TF-1 cellswere cultured for 1 hr with 20 μg/ml of IL-4 receptor alpha-specificmuteins or with negative control muteins. Further aliquots of cells werecultured with medium alone for 1 hr at 37° C. in a humidified atmosphereof 5% CO₂ in air. Subsequently, human recombinant IL-4 or IL-13 wasadded at a final concentration of 0.8 ng/ml or 12 ng/ml respectively andthe cultures were incubated for 10 min at 37° C. in a humidifiedatmosphere of 5% CO₂ in air.

Cells were fixed for 10 min at room temperature (RT) by the addition of42 μl of 37% formaldehyde (1.5% final concentration) and transferred to5 ml round bottomed polystyrene tubes (BD Falcon). Cells were washedwith 2 ml PBS containing 1% FCS (PBS-FCS), pelleted by centrifugation at1200 rpm for 5 min and the supernatant was discarded. Cells werepermeabilized by the addition of 500 μl ice-cold methanol whilstvortexing vigorously. After 10 min incubation at 4° C. the cells werewashed twice by centrifugation at 1200 rpm for 5 min with 2 ml ofPBS-FCS. The cells were resuspended in 100 μl of PBS-FCS and stainedwith 20 μl of anti-phosphorylated STAT-6 phycoerythrin (PE)-labelledantibody (clone Y641; BD Biosciences) for 30 min at RT protected fromlight. Finally, the cells were washed twice with 2 ml of PBS-FCS bycentrifugation at 1200 rpm for 5 min and resuspended in 500 μl ofPBS-FCS. The cells were analyzed by flow cytometry using a FACScaliburcytometer (BD Biosciences). Data were collected from at least 10000gated cells.

The ability of the IL-4 receptor alpha-specific muteins S191.4 B24 (SEQID NO: 5) and S191.4 K19 (SEQ ID NO: 6) to inhibit IL-4 and IL-13mediated STAT-6 phosphorylation in TF-1 cells was measured by flowcytometry. A gate was set on intact cells to exclude 99% of the controlunstained population on the basis of FL2 values (channel 2 fluorescence;PE intensity) using control TF-1 cells (unstimulated and unstained) onthe basis of cell size (forward scatter; FSC) and cell granularity (sidescatter; SSC). A further aliquot of unstimulated cells was stained withanti-phosphorylated STAT-6 PE-labelled antibody.

Results of the STAT-6 phosphorylation assay clearly show that the IL-4receptor alpha-specific muteins S191.4 B24 and S191.4 K19 markedlyinhibit IL-4 and IL-13 induced STAT-6 phosphorylation in TF-1 cells(data summarized in Table III).

TABLE III Ability of S191.4 B24 and S191.4 K19 (SEQ ID NO: 5 and 6) toinhibit STAT-6-phosphorylation-induced in TF-cells by IL-4 and IL-13 wasmeasured by flow cytometry. The percentage of gated cells stainingpositive for STAT-6 phosphorylation and the median fluorescenceintensity (MFI) of all gated cells are depicted. Treatment % PositiveMFI Unstained 1 3.8 Stained unstimulated 6 5.8 IL-4 75 15.8 IL-13 7716.4 pTLPC10 + IL-4 (neg control) 72 13.1 pTLPC10 + IL-13 (neg control)84 18.6 S191.4 K19 + IL-4 6 4.9 S191.4 K19 + IL-13 8 5.0 S191.4 B24 +IL-4 6 4.8 S191.4 B24 + IL-13 11 5.5

Example 12 Anti-Human IL-4 Receptor Alpha Muteins are Cross-ReactiveAgainst Cynomolgus Peripheral Blood Lymphocytes

Whole blood from healthy human volunteers was collected by the clinicalpharmacology unit (CPU) at Astra Zeneca (Macclesfield, UK) in 9 mllithium-heparin tubes. Samples of heparinized whole blood fromcynomolgus (pooled from a minimum of two animals) were obtained fromHarlan Sera-Lab (Bicester, UK) or B and K Universal Ltd (Hull, UK).

Human and cynomolgus whole blood was diluted 1:5 with erythrocyte lysisbuffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM EDTA, pH 7.2-7.4) andfollowing inversion incubated at room temperature for 10 min. Cells werecentrifuged at 1200 rpm for 5 min and supernatant removed. Cells wereresuspended in lysis buffer and the procedure repeated until thesupernatant no longer contained hemoglobin. Cells were re-suspended inthe same volume of freezing medium (1:10, dimethyl sulfoxide:fetal calfserum) as the original volume of blood and transferred to cryogenicvials. Each vial contained the cells from 1 ml of blood. Cells werefrozen overnight at −80° C. and transferred to liquid nitrogen forstorage.

Frozen peripheral blood cells were rapidly thawed at 37° C. and washedwith FACS buffer (PBS/1% FCS). Cell pellets were re-suspended in FACSbuffer (1 ml buffer/vial). 100 μl aliquots were placed into 96 wellround-bottomed plates, 100 μl of FACS buffer added per well, the platescentrifuged at 1200 rpm for 5 min at 4° C. and the supernatantdiscarded. Subsequently, cells were resuspended by vortexing at lowspeed and 100 μl of diluted primary antibody (anti-CD124 or IgG1 isotypecontrol, eBioscience, 10 μg/ml) or anti-IL-4 receptor alpha muteins (10μg/ml) were added and cells were incubated on ice for 30 min. Cells werewashed once by the addition of 100 μl FACS buffer and centrifugation at1200 rpm for 5 min at 4° C., the supernatant was discarded and the cellswere resuspended by vortexing at low speed. This was repeated twice moreusing 200 μl of FACS buffer to wash cells. After the finalcentrifugation the cell pellet was re-suspended in 100 μl of theappropriate secondary antibody at 5 μg/ml (biotinylated anti-humanlipocalin-1 antibody (R&D Systems) or biotinylated rat anti-mouse IgG(Insight Biotechnology Ltd)) and cells were incubated on ice for 30 min.Cells were washed once in 100 μl of FACS buffer by centrifugation at1200 rpm for 5 min at 4° C., the supernatant discarded and cellsresuspended by vortexing at low speed. Two further washes were performedusing 200 μl of FACS buffer and centrifugation at 1200 rpm for 5 min at4° C. After the final centrifugation the cell pellet was re-suspended in100 μl of the detection reagent (phycoerthyrin [PE]-labelledstreptavidin (eBioscience); 1.25 μg/ml) and incubated for 30 min on icein the dark. After three further wash steps as before, the cells weretaken up in 200 μl FACS buffer, transferred into 40×6 mm test tubes andanalyzed by flow cytometry using a FACScalibur cytometer. Control cellswere unstained. Using the unstained control cells, an intact lymphocytecell gate was set on cell size (forward scatter; FSC) and cellgranularity (side scatter; SSC) (Chrest, F. J. et al. (1993).Identification and quantification of apoptotic cells following anti-CD3activation of murine G0 T cells. Cytometry 14: 883-90). This region wasunaltered between samples analyzed on the same day. A marker was drawnto discriminate between IL-4 receptor alpha⁺ and IL-4 receptor alphapopulations, based on FL2 (channel 2 fluorescence; PE intensity) valuesin the control unstained population; marker 1 (M1) IL-4Rα⁺ cells was seton the basis of exclusion of 99% of the unstained population. For eachsample, data from at least 1×10⁴ cells were acquired.

Muteins S191.5 K12, S.148.3 J14-AM2C2, S.191.4 B24, S.191.4 K19, andS.197.8 D22 (SEQ ID NOs: 3-6 and 8) displayed high levels of binding tocynomolgus lymphocytes, IL-4 receptor alpha⁺ cells varied between 61%and 80% and MFI values varied between 6.0 and 9.2 (Table 2). VariantS.191.5 H16 (SEQ ID NO: 7) also specifically binds to cynomolguslymphocytes but with reduced affinity compared to the remaining muteins(41% IL-4 receptor alpha⁺ cells; MFI values 4.1).

In parallel, the ability of these IL-4 receptor alpha-specific muteinsto bind to peripheral blood lymphocytes from one human donor was alsoanalyzed by flow cytometry. All anti-IL-4 receptor alpha muteinsexhibited considerably higher levels of binding to human cells thanthose observed for the pTLPC10 negative control. IL-4 receptor alpha⁺cells varied between 60% and 76% and MFI values varied between 7.4 and9.7. Cells stained with pTLPC10 negative control displayed low levels ofnonspecific binding, with 9% cells recorded as IL-4 receptor alpha⁺ withMFI values of 3.2. Muteins S191.5 K12, S.191.4 B24, and S.191.4 K19 (SEQID NOs: 3, 5 and 6) displayed similar binding affinity to peripheralblood lymphocyte of a second human donor (data not shown).

TABLE IV Ability of IL-4 receptor alpha-specific muteins to bind humanand cynomolgus peripheral blood lymphocytes, analyzed by flow cytometry.The percentage of gated cells staining positive for IL-4 receptor alphaand the median fluorescence intensity (MFI) of all gated cells areshown. Human Cynomolgus peripheral blood peripheral cells blood cellsTreatment % Positive MFI % Positive MFI Unstained 1 2.4 1 1.7 pTLPC10(neg control) 9 3.2 5 1.9 S.191.4 K19 72 8.9 65 6.6 S.191.5 K12 74 9.778 9.0 S.191.4 B24 74 9.3 80 9.2 S.148.3 J14-AM2C2 76 9.6 68 6.8 S.191.5H16 72 9.0 42 4.1 S.197.8 D22 72 9.3 70 7.1

Example 13 Phagemid Presentation and Selection of Tlc Muteins withAffinity for Human VEGF

Phagemid display and selection employing the phagemids obtained fromExample 1 was performed essentially as described in Example 2 with thefollowing modifications: The target protein, i.e. a recombinant fragmentof human VEGF-A (VEGF₈₋₁₀₉, amino acids 8-109 of the mature polypeptidechain) was employed at a concentration of 200 nM and was presented tothe phagemid library as biotinylated protein with subsequent capture ofthe phage-target complex using streptavidin beads (Dynal) according tothe instructions of the manufacturer. Four rounds of selection wereperformed.

The target protein was obtained by introducing the nucleic acids codingfor amino acids 8 to 109 of the mature polypeptide chain of human VEGF A(SWISS PROT Data Bank Accession No. P15692) into the expression vectorpET11c (Novagen). Therefore, BamHI and NdeI restriction sites wereintroduced at the 3′ and the 5′ end of the cDNA of the human VEGFfragment, respectively, and used for subcloning of the VEGF genefragment.

E. coli BL21(DE3) was transformed with the resulting expression plasmidand cytoplasmic production of VEGF₈₋₁₀₉ was achieved after induction ofan expression culture in ampicillin-containing LB medium with IPTG for 3h at 37° C. After centrifugation at 5000 g for 20 min the cell pelletwas resuspended in 200 ml PBS for each 2 l of culture broth and againcentrifuged at 5000 g for 10 min prior to incubation at −20° C. overnight. Each cell pellet obtained from 500 ml culture broth wasresuspended in 20 ml 20 mM Tris-HCl (pH 7.5), 5 mM EDTA and sonificatedon ice, four times for 10 seconds. After centrifugation for 10 min with10000 g at 4° C., inclusion bodies were solubilized with 15 mlpre-chilled IB buffer (2 M urea, 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl),sonificated and centrifuged as above. Afterwards, the cell pellets weresolubilized with 20 ml IB buffer and again centrifuged like above priorto solubilization in 25 ml solubilization buffer (7.5 M urea, 20 mMTris-HCl (pH 7.5), 4 mM DTT). The cell suspension was stirred for 2 h atambient temperature, centrifuged at 40000 g for 15 min at 4° C. and thesupernatant containing the recombinant VEGF was filtrated (0.45 μm).Refolding was achieved by dialysis (3.5 kDa molecular weight cut-off) atambient temperature over night against 51 buffer 1 (20 mM Tris-HCl (pH8.4), 400 mM NaCl, 1 mM Cystein) followed by dialysis against 5 l buffer2 (20 mM Tris-HCl (pH 8.4), 1 mM Cystein) and 2 subsequent dialysissteps with 5 l buffer 3 (20 mM Tris-HCl (pH 8.4)). After centrifugation(40000 g, 20 min, 4° C.) and concentration the recombinant VEGF fragmentwas purified according to standard methodologies by subsequent ionexchange chromatograpy (Q-Sepharose) and size exclusion chromatography(Superdex 75).

Example 14 Identification of VEGF-Binding Muteins Using aHigh-Throughput ELISA Screen

Screening of the Tlc muteins obtained in Example 13 was performedessentially as described in Example 3 with the modification that therecombinant target protein VEGF₈₋₁₀₉ obtained from Example 11 wasemployed at 5 μg/ml and was directly coated to the microtitre plate.Screening of altogether 2124 clones lead to the identification of 972primary hits indicating that successful isolation of muteins from thelibrary had taken place. Using this approach the Tlc mutein 5168.4-L01(SEQ ID NO:26) was identified.

Example 15 Affinity Maturation of Tlc Mutein S168.4-L01 UsingError-Prone PCR

Generation of a library of variants based on mutein S168.4-L01 wasperformed essentially as described in Example 4 using theoligonucleotides TL50 bio (SEQ ID NO:15) and TL51 bio (SEQ ID NO:16)resulting in a library with 5 substitutions per structural gene onaverage.

Phagemid selection was carried out as described in Example 13 usinglimited target concentration (10 nM, 1 nM and 0.2 nM VEGF₈₋₁₀₉), orshort incubation times (1 and 5 minutes) with and without limitingtarget concentrations (10 nM, 100 nM). Four rounds of selection wereperformed.

Example 16 Affinity Screening of VEGF-Binding Muteins Using aHigh-Throughput ELISA Screen

Screening of the muteins selected in Example 15 was performed asdescribed in Example 14 with the modification that a monoclonal anti-T7tag antibody (Novagen) was coated onto the ELISA plate in order tocapture the produced muteins and binding of biotinylated VEGF₈₋₁₀₉ (500nM and 50 nM) to the captured Tlc muteins was detected usingHRP-conjugated Extravidin.

A large number of clones were identified having improved affinity ascompared to the mutein 5168.4-L01, which served as the basis foraffinity maturation. Using this approach clones 5209.2-C23, 5209.2-D16,5209.2-N9, S209.6-H7, S209.6-H10, 5209.2-M17, 5209.2-010 (SEQ IDNOs:27-33) were identified.

Example 17 Production of VEGF Binding Muteins

Production was performed essentially as described in Example 7.

Example 18 Affinity Determination of VEGF-Specific Muteins EmployingBiacore

Affinity measurements were performed essentially as described in Example8 with the modification that approximately 250 RU of recombinant VEGFwas directly coupled to the sensor chip using standard amine chemistry.40 μl of the Tlc muteins obtained from Example 15 was injected at aconcentration of 400 nM.

Results from the affinity determinations of the muteins 5209.2-C23,S209.2-D16, 5209.2-N9, 5209.6-H7, 5209.6H10, 5209.2-M17 and S209.2-O10(SEQ ID NOs:27-33) are summarized in Table V.

TABLE V Affinities of selected muteins of the invention for VEGF asdetermined by Biacore measurements at 25° C. k_(on) k_(off) AffinityClone [10⁴ 1/Ms] [10⁻⁵ 1/s] [nM] S209.2-C23 3.6 1.3 0.37 S209.2-D16 3.83 0.79 S209.2-N9 5.9 7.1 1.2 S209.6-H7 6.4 4.4 0.68 S209.6-H10 4.6 4.40.97 S209.2-M17 2.8 2.0 0.72 S209.2-O10 3.2 0.67 0.21

Example 19 Identification of Antagonists of VEGF Using an InhibitionELISA

Inhibition of the interaction between VEGF and VEGF Receptor 2 (VEGF-R2)was evaluated in an inhibition ELISA. To this end, a constantconcentration of biotinylated VEGF₈₋₁₀₉ (1 nM) was incubated with adilution series of the respective Tlc mutein and the amount of VEGF withan unoccupied VEGF-R2 binding site was quantified in an ELISA where ananti-VEGF antibody interfering with the VEGF/VEGF-R2 interaction(MAB293, R&D Systems) had been coated. Bound VEGF was detected usingHRP-conjugated Extravidin (Sigma) and compared to a standard curve ofdefined amounts of VEGF. Results from measurements employing muteins5209.2-C23, 5209.2-D16, 5209.2-N9, 5209.6-H7, 5209.6-H10, 5209.2-M17 andS209.2-O10 (SEQ ID NOs:27-33) are summarized in Table VI.

TABLE VI Antagonistic ability and affinities for VEGF of selected tearlipocalin muteins of the invention as determined by competition ELISA.Affinity Competition ELISA Clone Ki [nM] S209.2-C23 2.3 S209.2-D16 3.9S209.2-N9 2.8 S209.6-H7 2.4 S209.6-H10 1.3 S209.2-M17 2.0 S209.2-O100.83

Example 20 Identification of VEGF Antagonists Using a HUVECProliferation Assay

Inhibition of VEGF and FGF-2 stimulated HUVEC cell proliferation wasassessed essentially as previously described (Korherr C., Gille H,Schafer R., Koenig-Hoffmann K., Dixelius J., Egland K. A., Pastan I. &Brinkmann U. (2006) Proc. Natl. Acad. Sci (USA) 103(11) 4240-4245) withthe following modifications: HUVEC cells (Promocell) were grownaccording to the manufacturer's recommendations and used between passage2 and 6. On day one, 1.400 cells were seeded in complete medium(Promocell). On the following day, cells were washed and basal mediumcontaining 0.5% FCS, hydrocortisone and gentamycin/amphotericin but noother supplements (Promocell) was added. VEGF-specific mutein5209.2-C23, S209.2-D16, 5209.2-N9, 5209.6-H7, 5209.6-H10, 5209.2-M17,S209.2-O10 (SEQ ID NOs:27-33), wildtype tear lipocalin (gene product ofpTLPC10; as control) or VEGF-specific therapeutic monoclonal antibodyAvastin® (Roche; as control) was added in a dilution series at theindicated concentration in triplicate wells and after 30 min eitherhuman VEGF165 (R&D Systems) or human FGF-2 (Reliatech) was added.Viability of the cells was assessed after 6 days with CellTiter 96Aqueous One (Promega) according to the manufacturer's instructions.Results from measurements employing muteins 5209.2-C23, S209.2-D16,5209.2-N9, S209.6-H7, 5209.6-H10, 5209.2-M17 and S209.2-O10 (SEQ IDNOs:27-33) are shown in FIG. 21. All muteins of the invention showmarked inhibition of VEGF-induced proliferation of HUVEC cells, which iscomparable to or better than the Avastin®-induced inhibition, whereaswildtype tear lipocalin does not inhibit VEGF-induced cellproliferation. FGF-2-induced cell proliferation is not affected by anyof the VEGF-specific muteins, TLPC10 or Avastin® (not shown).

Example 21 Phagemid Presentation and Selection of Tlc Muteins AgainstVEGF-R2

Phagemid display and selection employing the phagemids obtained fromExample 1 was performed essentially as described in Example 2 with thefollowing modifications: Target protein VEGF-R2-Fc (R&D Systems) wasemployed at a concentration of 200 nM and was presented to the libraryas Fc-fusion protein with subsequent capture of the phage-target complexusing protein G beads (Dynal) according to the instructions of themanufacturer. Four rounds of selection were performed.

Example 22 Identification of VEGF-R2-Binding Muteins Using aHigh-Throughput ELISA Screen

Screening was performed essentially as described in Example 3 with themodification that the target protein VEGF-R2-Fc (R&D Systems) was usedat a concentration of 2.5 μg/ml.

Screening of 1416 clones, obtained from the procedure described underExample 21 lead to the identification of 593 primary hits indicatingthat successful isolation of muteins from the library of the inventionhad taken place. Using this approach the mutein S175.4 H11 (SEQ IDNO:34) was identified.

Example 23 Affinity Maturation of VEGF-R2-Specific Mutein 5175.4 H11Using Error-Prone PCR

Generation of a library of variants based on the mutein S175.4 H11 wasperformed essentially as described in Example 4 using theoligodeoxynucleotides TL50 bio (SEQ ID NO:15) and TL51 bio (SEQ IDNO:16) resulting in a library with 2 substitutions per structural geneon average.

Phagemid selection was carried out as described in Example 21 usinglimited target concentration (5 nM, 1 nM and 0.2 nM of VEGF-R2-Fc),extended washing times (1 h) in the presence of competing recombinantVEGF₈₋₁₀₉ (100 nM) or short incubation times (2 and 5 minutes) with andwithout limiting target concentrations (10 nM, 100 nM). Four rounds ofselection were performed.

Example 24 Affinity Screening of VEGF-R2-Binding Muteins Using aHigh-Throughput ELISA Screen

Screening was performed as described in Example 3 with the modificationthat monoclonal anti-T7 tag antibody (Novagen) was coated onto the ELISAplate in order to capture the produced Tlc muteins and binding ofVEGF-R2-Fc (R&D Systems, 3 nM and 1 nM) to the captured muteins wasdetected using a HRP-conjugated antibody against the Fc domain ofVEGF-R2-Fc.

A large number of clones were identified having improved affinitycompared to the muteins S175.4 H11, which served as the basis foraffinity maturation. Using this approach the clones S197.7-N1,S197.2-I18, S197.2-L22, S197.7-B6 and S197.2-N24 (SEQ ID NOs:35-39) wereidentified.

Example 25 Production of VEGF-R2 Binding Muteins

Production was performed essentially as described in Example 7.

Example 26 Affinity Determination of VEGF-R2-Specific Muteins UsingBiacore

Affinity measurements were performed essentially as described in Example8 with the modifications that approximately 500 RU of VEGF-R2-Fc (R&DSystems) was captured and 80 μl of mutein was injected at aconcentration of 1.5 μM.

Results from the measurements employing S175.4-H11, S197.7-N1,S197.2-I18, S197.2-L22, 5197.7-B6 and 5197.2-N24 (SEQ ID NOs:35-39) aresummarized in Table VII.

TABLE VII Affinities of selected muteins of the invention for VEGF-R2 asdetermined by Biacore measurements. k_(on) k_(off) Affinity Clone [10⁴1/Ms] [10⁻⁵ 1/s] [nM] S175.4-H11 0.9 36 35 S197.7-N1 2.1 11 5.5S197.2-I18 2.7 8.3 3.1 S197.2-L22 1.2 2.4 3.3 S197.7-B6 2.3 13 6S197.2-N24 2.4 6.4 2.7

Example 27 Identification of Antagonists of VEGF Using an InhibitionELISA

Inhibition of the interaction between VEGF and VEGF-R2 by theVEGF-R2-specific muteins was evaluated in an inhibition ELISA.Therefore, a constant concentration of VEGF-R2 (4 nM VEGF-R2-Fc, R&DSystems) was incubated with a dilution series of the respective muteinand the amount of VEGF-R2 with an unoccupied VEGF binding site wasquantified in an ELISA where VEGF₈₋₁₀₉ had been coated. Bound VEGF-R2was detected using HRP-conjugated anti-human Fc antibody (Dianova) andcompared to a standard curve of defined amounts of VEGF-R2-Fc. Resultsfrom measurements of 5175.4-H11, S197.7-N1, S197.2-I18, S197.2-L22,S197.7-B6 and S197.2-N24 (SEQ ID NOs:35-39) are summarized in TableVIII.

TABLE VIII Antagonistic ability and affinities for VEGF-R2 of selectedtear lipocalin muteins of the invention as determined by competitionELISA. Affinity competition ELISA Clone Ki [nM] S175.4-H11 12.9S197.7-N1 12 S197.2-I18 5.5 S197.2-L22 3.5 S197.7-B6 3.8 S197.2-N24 2.3

Example 28 Site-Specific Modification of IL-4 Receptor Alpha-SpecificMuteins with Polyethylene Glycol (PEG)

An unpaired cysteine residue was introduced instead of the amino acidGlu at position 131 of the IL-4 receptor alpha-specific mutein S148.3J14 (SEQ ID NO:2) by point mutation in order to provide a reactive groupfor coupling with activated PEG. The recombinant mutein carrying thefree Cys residue was subsequently produced in E. coli as described inExample 7.

For coupling of the mutein S148.3 J14 with PEG, 5.1 mg polyethyleneglycol maleimide (average molecular weight 20 kDa, linear carbon chain;NOF) was mixed with 3 mg of the protein in PBS and stirred for 3 h atambient temperature. The reaction was stopped by the addition ofbeta-mercaptoethanol to a final concentration of 85 μM. After dialysisagainst 10 mM Tris-HCl (pH 7.4), the reaction mixture was applied to aHiTrap Q-XL Sepharose column (Amersham) and the flow-through wasdiscarded. The PEGylated mutein was eluted and separated from unreactedprotein applying a linear salt gradient from 0 mM to 100 mM NaCl.

Example 29 Affinity Measurement of the PEGylated Mutein 5148.3 J14 UsingBiacore

Affinity measurements were performed essentially as described in Example8 with the modifications that approximately 500 RU of IL-4 receptoralpha-Fc (R&D Systems) was immobilized and 80 μl of the purifiedPEGylated mutein was injected at concentrations of 200 nM, 67 nM and 22nM. The result of the measurement is depicted in FIG. 22 and summarizedin Table IX. The affinity of the mutein S148.3 J14 in its PEGylated form(ca. 30 nM) is almost unchanged as compared to the non-PEGylated mutein(ca. 37 nM, cf. Example 8).

TABLE IX Affinity of the PEGylated mutein of the invention S148.3 J14for IL-4 receptor alpha as determined by Biacore. k_(on) k_(off)Affinity Clone [10⁵ 1/Ms] [10⁻³ 1/s] [nM] S148.3 J14-PEG 1.64 4.93 30

Example 30 Affinity Maturation of the Mutein S209.6-H10 Using aSite-Directed Random Approach

A library of variants based on the mutein 5209.6-H10 (SEQ ID NO:30) wasdesigned by randomization of the residue positions 26, 69, 76, 87, 89and 106 to allow for all 20 amino acids on these positions. The librarywas constructed essentially as described in Example 1 with themodification that the deoxynucleotides TL107 (covering position 26),TL109 (covering positions 87 and 89), TL110 (covering position 106) andTL111 (covering positions 69 and 76) were used instead of TL46, TL47,TL48 and TL49, respectively. Phagemid selection was carried outessentially as described in Example 13 using either limited targetconcentration (10 pM and 2 pM and 0.5 pM of VEGF₈₋₁₀₉) or combined witha competitive monoclonal antibody against VEGF (Avastin®). Four roundsof selection were performed.

TL107  (SEQ ID NO: 40) GAAGGCCATGACGGTGGACNNSGGCGCGCTGAGGTGCCTC TL109 (SEQ ID NO: 41) GGCCATCGGGGGCATCCACGTGGCANNSATCNNSAGGTCGCACGTGAAG GACTL110  (SEQ ID NO: 42) CACCCCTGGGACCGGGACCCCSNNCAAGCAGCCCTCAGAG TL 111 (SEQ ID NO: 43) CCCCCGATGGCCGTGTASNNCCCCGGCTCATCAGTTTTSNNCAGGACGGCCCTCACCTC

Example 31 Affinity Screening of VEGF-Binding Muteins UsingHigh-Throughput ELISA Screening

Screening was performed as described in Example 14 with the modificationthat a concentration of 1 μg/m1VEGF was used and the additions that

-   -   i) a monoclonal anti-T7 tag antibody (Novagen) was coated onto        the ELISA plate in order to capture the produced muteins and        binding of biotinylated VEGF (3 nM and 1 nM) to the captured        muteins of tear lipocalin was detected using a HRP (horseradish        peroxidase)-conjugated extravidin. Additionally, in alternative        screening setups    -   ii) instead of human VEGF₈₋₁₀₉ mouse VEGF₁₆₄ (R&D Systems) was        directly coated to the microtiter plate (1 μg/m1).    -   iii) the extract containing the VEGF-binding muteins was heated        to 60° C. for 1 hour.    -   iv) mAB293 (R&D Systems, 5 μg/ml) was coated onto the ELISA        plate and biotinylated VEGF₈₋₁₀₉ was preincubated with the        expressed muteins. Binding of VEGF₈₋₁₀₉ to mAB293 was detected        using HRP (horseradish peroxidase)-conjugated extravidin.

A large number of clones were identified having improved affinity ascompared to the mutein 5209.6-H10, which served as the basis foraffinity maturation. Using this approach clones S236.1-A22, S236.1-J20,S236.1-M11 and S236.1-L03 (SEQ ID NOs:44-47) were identified.

In this context it is noted that due to the deletion of the first 4amino acids of tear lipocalin in the muteins of the invention, the aminoacid sequence is depicted starting from sequence position 5 (alanine) ofthe deposited wild type tear lipocalin sequence of tear lipocalin, sothat Ala5 is depicted as N-terminal amino acid. In addition, theC-terminal amino acid Asp158 of the wild type tear lipocalin is replacedby an alanine residue (residue 154 in SEQ ID NO: 44-47, see also theother muteins of the invention such as SEQ ID NO: 26-40). Furthermore,the amino acid sequence of muteins 5236.1-A22, 5236.1-J20, 5236.1-M11and 5236.1-L03 together with the STREP-TAG® II that is fused to theC-terminus of tear lipocalin for the construction of the nave library ofExample 1 is shown in SEQ ID NO:52 (S236.1-A22-strep), SEQ ID NO: 53(S236.1-J20-strep), SEQ ID NO: 54 (S236.1-M11-strep) and SEQ ID NO: 55(S236.1-L03-step). Also this illustrates the variability of the sequenceof tear lipocalin muteins of the invention apart from the indicatedmutated positions/mutations that are necessary to provide the respectivemutein with the ability to specifically bind the given target such asVEGF, or VEGF-R2 or interleukin 4 receptor alpha chain (IL-4 receptoralpha).

Example 32 Production of VEGF Binding Muteins

Production was performed essentially as described in Example 7.

Example 33 Affinity Determination of VEGF-Specific Muteins EmployingBiacore

Affinity measurements were performed essentially as described in Example18. (See also FIG. 23 in which Biacore measurements of the binding ofhuman tear lipocalin mutein 5236.1-A22 (SEQ ID NO:44) to immobilizedVEGF₈₋₁₀₉ are illustrated). Briefly, VEGF₈₋₁₀₉ was immobilized on a CMSchip using standard amine chemistry. Lipocalin mutein was applied with aflow rate of 30 μl/min at six concentrations from 500 nM to 16 nM.Evaluation of sensorgrams was performed with BIA T100 software todetermine Kon, Koff and KD of the respective muteins.

TABLE X Affinities of selected muteins of the invention for VEGF asdetermined by Biacore measurements at 25° C. k_(on) k_(off) AffinityMutein [10⁴ 1/Ms] [10⁻⁵ 1/s] [nM] S236.1-A22 8.8 2.2 0.25 S236.1-J20 7.92.2 0.28 S236.1-L03 6.8 4.4 0.64 S236.1-M11 7.3 2.3 0.31

Example 34 Identification of Antagonists of VEGF Using an InhibitionELISA

Inhibition of the interaction between VEGF and VEGF Receptor 2 (VEGF-R2)was evaluated in an inhibition ELISA essentially as described in Example19 with the modification that the incubation time of 1 hour was reducedto 10 minutes. Inhibition constants are summarized in the followingTable:

TABLE XI Antagonistic ability and affinities for VEGF of selected tearlipocalin muteins of the invention as determined by competition ELISA.Affinity Competition ELISA Mutein Ki [nM] S236.1-A22 5.8 S236.1-J20 6.3S236.1-L03 9.4 S236.1-M11 6.4

Example 35 Determination of Cross-Reactivity of VEGF-Specific MuteinsS236.1-A22 Using Biacore

Affinity measurements were performed essentially as described in Example18 with the modification that mutein S236.1-A22 (SEQ ID NO:44) wasimmobilized on the sensor chip. 70 μl of sample was injected at aconcentration of 250 nM.

The qualitative comparison of the results as shown in FIG. 24 illustratethat the truncated form hVEGF₈₋₁₀₉ and hVEGF₁₂₁ show basically identicalsensorgrams indicating similar affinity to the tear lipocalin muteinS236.1-A22 (SEQ ID NO:44). The splice form hVEGF₁₆₅ also shows strongbinding to the lipocalin mutein, while the respective mouse orthologmVEGF164 has slightly reduced affinity. Isoforms VEGF-B, VEGF-C andVEGF-D and the related protein PlGF show no binding in this experiment(data not shown).

Example 36 Determination of Thermal Denaturation for VEGF-BindingMuteins by Use of CD Spectroscopy

Circular dichroism measurements were performed essentially as describedin Example 14 of the International patent application WO2006/056464,with the modification that the wavelength used was 228 nM. The meltingtemperature T_(m) of the tear lipocalin mutein 5236.1-A22 (SEQ ID NO:44)for example was determined to be 75° C.

Example 37 Stability Test of S236.1-A22

Stability of VEGF-binding mutein S236.1-A22 at 37° C. in PBS and humanserum was tested essentially as described in Example 15 of theInternational patent application WO2006/056464 except that theconcentration utilized was 1 mg/ml. No alteration of the mutein could bedetected during the seven day incubation period in PBS as judged byHPLC-SEC (data not shown). Incubation of the lipocalin mutein in humanserum resulted in a drop of affinity after 7 days to approx. 70%compared to the reference (See also FIG. 25A).

Example 38 Fusion of Anti-VEGF Muteins with an Albumin-Binding Domain

For serum half-life extension purposes anti-VEGF muteins wereC-terminally fused with an albumin-binding domain (ABD). The geneticconstruct used for expression is termed pTLPC51 S236.1-A22 (SEQ IDNO:50). (See FIG. 26)

The preparative production of VEGF-specific mutein-ABD fusions orTlc-ABD (as control) was performed essentially as described in Example7.

Affinity measurements using surface plasmon resonance (Biacore) wereperformed essentially as described in Example 18. The affinity of theABD-fusion of tear lipocalin mutein S236.1-A22 (A22-ABD) (SEQ ID NO: 51)(200 pM) towards recombinant VEGF was found basically unaltered andmeasured to be 260 pM (see FIG. 27).

Additionally, the integrity of the ABD-domain was tested by the samemethod, as described in Example 8, with the modification thatapproximately 850 RU of human serum albumin was directly coupled to thesensor chip using standard amine chemistry. 60 μl of mutein-ABD fusions(A22-ABD (SEQ ID NO: 51) or wildtype Tlc-ABD (SEQ ID NO:49)) wereinjected at a concentration of 500 nM. Their affinity was measured to beapprox. 20 nM

The stability of the ABD-fusion of S236.1-A22 (SEQ ID NO: 51) in humanserum was tested essentially as described in Example 37. No loss ofactivity could be detected during the seven day incubation period. (SeeFIG. 25B)

The functionality of the lipocalin mutein A22-ABD (ABD-fusion ofS236.1-A22) in the presence of human serum albumin was tested by itsability to inhibit VEGF induced HUVEC proliferation. The assay wasperformed as described in Example 39 except that human serum albumin(HSA, 5 μM) was added where indicated. At 5 μM HSA, >99.8% of A22-ABD isassociated with HSA at any given time due to the nanomolar affinity ofA22-ABD for HSA (see FIG. 28). IC50 values were determined to be asfollows:

S236.1-A22-ABD IC50: 760 pM S236.1-A22-ABD (+HSA) IC50: 470 pM

Example 39 Inhibition of VEGF Induced HUVEC Proliferation

HUVEC (Promocell) were propagated on gelatine-coated dishes and usedbetween passages P2 and P8. On day 1, 1400 cells were seeded per well ina 96 well plate in complete medium. On day 2, cells were washed and 100μl of basal medium containing 0.5% FCS, hydrocortisone andgentamycin/amphotericin was added. Proliferation was stimulated with 20ng/ml VEGF165 or 10 ng/ml FGF-2 which were mixed with the lipocalinmutein S236.1-A22 (SEQ ID NO:44), incubated for 30 min and added to thewells. Viability was determined on day 6, the results are expressed as %inhibition. IC50 values were determined to be as follows (see also FIG.29).

S236.1-A22 IC50: 0.51 nM Avastin IC50: 0.56 nM

FGF-2 mediated stimulation was unaffected by VEGF antagonists (data notshown).

Example 40 Inhibition of VEGF-Mediated MAP Kinase Activation in HUVEC

HUVEC were seeded in 96-well plates at 1,400 cells per well in standardmedium (Promocell, Heidelberg). On the following day, FCS was reduced to0.5% and cultivation was continued for 16 h. Cells were then starved in0.5% BSA in basal medium for 5 h. HUVEC were stimulated with VEGF₁₆₅(Reliatech, Braunschweig) for 10 min in the presence of increasingconcentrations of tear lipocalin mutein A22 or Avastin (bevacizumab,Genentech/Roche) in order to obtain a dose-response curve.Phosphorylation of the MAP kinases ERK1 and ERK2 was quantified using anELISA according to the manufacturer's manual (Active Motif, Rixensart,Belgium). The IC 50 value was determined to be 4.5 nM for the mutein A22(SEQ ID NO:44) and 13 nM for Avastin® (see FIG. 30).

Example 41 Vascular Permeability Assay with Local Administration of TearLipocalin Mutein

Duncan-Hartley guinea pigs weighing 350±50 g were shaved on the shoulderand on the dorsum. The animals received an intravenous injection via theear vein of 1 ml of 1% Evan's Blue dye. Thirty minutes later 20 ngVEGF₁₆₅ (Calbiochem) was mixed with test substance or control article ata tenfold molar excess and injected intradermally on a 3×4 grid. Thirtyminutes later, animals were euthanized by CO₂ asphyxiation. One hourafter the VEGF injections, the skin containing the grid pattern wasremoved and cleaned of connective tissue. The area of dye extravasationwas quantified by use of an image analyzer (Image Pro Plus 1.3, MediaCybernetics) (see FIG. 31).

Example 42 CAM (Chick Chorioallantoic Membrane) Assay

Collagen onplants containing FGF-2 (500 ng), VEGF (150 ng) and tearlipocalin mutein (1.35 μg) or Avastin (10 μg) as indicated were placedonto the CAM of 10 day chicken embryos (4/animal, 10 animals/group). At24 h the tear lipocalin mutein or Avastin were reapplied topically tothe onplant at the same dose. After 72 h onplants were collected andimages were captured. The percentage of positive grids containing atleast one vessel was determined by a blinded observer. The medianangiogenic index is reported for the VEGF antagonists 5209.2-010 (SEQ IDNO:33) and Avastin® as well as wild type tear lipocalin control as thefraction of positive grids (see FIG. 32).

Example 43 Determination of Pharmacokinetic (PK) Parameters for A22 andA22-ABD in Mice

Pharmacokinetic (PK) parameters (half-life plasma concentration,bioavailibity) for tear lipocalin mutein 5236.1 A22 (SEQ ID NO:44) (4mg/kg) after i.v. and the fusion protein of muteiin 5236.1 A22 with ABD(SEQ ID NO:51) (5.4 mg/kg) following i.v. or i.p. single bolusadministration were determined in NMRI mice. Plasma was prepared fromterminal blood samples taken at pre-determined timepoints and theconcentrations of the lipocalin mutein were determines by ELISA. Resultswere analyzed using WinNonlin software (Pharsight Corp., Mountain View,USA). T_(1/2), A22 i.v.: 0.42 h; T_(1/2) A22-ABD i.v.: 18.32 h; T_(1/2),A22-ABD i.p.: 20.82 h. The bioavailability following i.p. administrationof the fusion protein A22-ABD was 82.5% (see FIG. 33).

Example 44 Vascular Permeability Assay with Systemic Administration ofTear Lipocalin Mutein

Twelve hours prior to the experiment, test substances or controls wereinjected intravenously into 3 animals per group. Group 1: PBS vehicle;Group 2: Avastin, 10 mg/kg; Group 3: mutein S236.1 A22-ABD, 6.1 mg/kg;Group 4: TLPC51: 6.1 mg/kg. At time=0 Evan's Blue was injected. Thirtyminutes later, 4 doses of VEGF (5, 10, 20 or 40 ng) were injectedintradermally in triplicate on a 3×4 grid. Thirty minutes after the VEGFinjections the animals were sacrificed and dye extravasation wasquantified as above (see FIG. 34).

Example 45 Tumor Xenograft Model

Irradiated (2.5 Gy, Co⁶⁰) Swiss nude mice were inoculated subcutaneouslywith 1×10⁷ A673 rhabdomyosarcoma cells (ATTC) in matrigel into the rightflank (n=12 per group). Treatments were administered intraperitoneallyand were initiated on the same day and continued for 21 days. Group 1:PBS vehicle, daily; Group 2: Avastin (bevacizumab, Genentech/Roche), 5mg/kg every 3 days; Group 3: mutein A22-ABD (SEQ ID NO:51), daily, 3.1mg/kg; Group 4: TLPC51, daily, 3.1 mg/kg. The dose of the lipocalinA22-ABD was chosen to achieve the constant presence of an equimolarnumber of VEGF binding sites of the mutein and Avastin based on theA22-ABD PK data and estimated serum half life of antibodies in mice.Tumor size was measured twice weekly with a calliper and the tumorvolume was estimated according to the formula (length×width²)/2. Micewere sacrificed when the tumor volume exceeded 2,000 mm³ (see FIG. 35).

Example 46 Screening of Lipocalin Mutein-Cys Variants

In order to provide a reactive group for coupling with e.g. activatedPEG, an unpaired cysteine residue was introduced by site-directedmutagenesis. The recombinant mutein carrying the free Cys residue wassubsequently produced in E. coli as described in Example 7, theexpression yield determined and the affinity measured by ELISAessentially as described in Example 14. Exemplary, results from theCys-screening of the VEGF-specific mutein S236.1-A22 (SEQ ID NO:44) aregiven in the table below. Cystein was introduced instead of the aminoacids Thr 40, Glu 73, Asp 95, Arg 90 and Glu 131 using the followingoligonucleotides

A22_D95C forward:  (SEQ ID NO: 56)GAGGTCGCACGTGAAGTGCCACTACATCTTTTACTCTGAGG, A22_D95C reverse: (SEQ ID NO: 57) CCTCAGAGTAAAAGATGTAGTGGCACTTCACGTGCGACCTC,A22_T40C forward:  (SEQ ID NO: 58)GGGTCGGTGATACCCACGTGCCTCACGACCCTGGAAGGG, A22_T40C reverse: (SEQ ID NO: 59) CCCTTCCAGGGTCGTGAGGCACGTGGGTATCACCGACCC,,A22_E73C forward:  (SEQ ID NO: 60)CCGTCCTGAGCAAAACTGATTGCCCGGGGATCTACACGG, A22_E73C reverse: (SEQ ID NO: 61) CCGTGTAGATCCCCGGGCAATCAGTTTTGCTCAGGACGG,A22_E131C forward:  (SEQ ID NO: 62) GCCTTGGAGGACTTTTGTAAAGCCGCAGGAG,A22_E131C reverse:  (SEQ ID NO: 63) CTCCTGCGGCTTTACAAAAGTCCTCCAAGGC,A22_R90C forward:  (SEQ ID NO: 64) CGTGGCAAAGATCGGGTGCTCGCACGTGAAGGACC, and A22_R90C reverse:  (SEQ ID NO: 65)GGTCCTTCACGTGCGAGCACCCGATCTTTGCCACG.

TABLE XII Affinity of the muteins S236.1-A22 and its Thr 40→ Cys (SEQ IDNO: 66), Glu 73→ Cys (SEQ ID NO: 67), Asp 95→ Cys (SEQ ID NO: 68), Arg90→ Cys (SEQ ID NO: 69), and Glu 131→ Cys (SEQ ID NO: 70) mutants forVEGF as determined by ELISA. Yield Affinity Clone [μg/L] [nM] S236.1-A221000 10 S236.1-A22 T40C 420 14 S236.1-A22 E73C 300 13 S236.1-A22 D95C750 10 S236.1-A22 R90C 470 10 S236.1-A22 E131C 150 >100

Example 47 Eotaxin-3 Secretion Assay

An Eotaxin-3 secretion assay was performed on A549 cells over 72 hours.Lung epithelial cells, such as A549 cells, secrete eotaxin-3 uponIL-4/IL-13 stimulation. Thus, A549 cells were treated with increasingconcentrations of the IL-4 receptor alpha binding mutein S191.4 B24 (SEQID NO:4) and stimulated with 0.7 nM IL-4 or 0.83 nM IL-13, respectively.Eotaxin-3 secretion was assessed after 72 hours using a commercialsandwich ELISA (R&D Systems). The results (FIG. 36) demonstrate that theIL-4 receptor alpha binding mutein S191.4 B24 inhibits IL-4 and IL-13mediated eotaxin-3 secretion in A549 cells with an IC₅₀ value of 32 and5.1 nM, respectively (Table XIII).

TABLE XIII IC₅₀ values of S191.4 B24 for IL-4 and IL-13 mediatedeotaxin-3 secretion in A549 cells. IC₅₀ (nM) IL-4 32 IL-13 5.1

Example 48 IL-4/IL-13 Mediated CD23 Induction on Peripheral BloodMononuclear Cells

Total human PBMCs were isolated from buffy coat. PBMCs were treated withincreasing concentrations of the IL-4 receptor alpha binding muteinS191.4 B24 and IL-4 or IL-13 were added to a final concentration of 1.0nM and 2.5 nM, respectively. PBMCs were cultured for 48 hours in RPMImedium containing 10% FCS. Cells were stained with anti-CD14-FITC andanti-CD23-PE antibodies and analyzed by flow cytometry. For each point,the percentage of double-positive cells out of all CD14 positivemonocytes was determined and plotted as a function of muteinconcentration.

From the obtained results, the IC₅₀ values of the mutein S191.4 B24 forinhibiting IL-4 and IL-13 mediated CD23 expression on monocytes wascalculated (Table XIV).

TABLE XIV IC₅₀ values of S191.4 B24 for IL-4 and IL-13 mediated CD23expression in PBMCs. IC₅₀ (nM) IL-4 905 IL-13 72

Example 49 Schild Analysis of the Affinity of the IL-4 Receptor AlphaBinding Mutein 5191.4 B24

A Schild analysis was carried out to confirm the hypothezisedcompetitive binding mode of the muteins and to determine the K_(d) oncells. TF-1 cells were treated with a fixed concentration of the IL-4receptor alpha binding mutein S191.4 B24 (0, 4.1, 12.3, 37, 111.1, 333.3or 1000 nM) and titrated with IL-4 and cell viability was assessed after4 days (FIG. 38A). EC₅₀ values were determined by non-linear regression.Traditional Schild analysis of the obtained results (FIG. 38B) yielded aKd of 192 pM (linear regression) and the more accurate non-linearregression yielded 116 pM. The Schild slope of 1.084 indicates acompetitive inhibition, i.e. the mutein and IL-4 compete for the IL-4receptor alpha binding.

Example 50 Picomolar Binding of the Mutein 5191.4 B24 to Primary B Cells

PBMCs were isolated from human blood and incubated with differentconcentrations of the IL-4 receptor alpha binding human tear lipocalinmutein S191.4 B24 or the wild-type human tear lipocalin (TLPC26). Cellswere then stained with anti-CD20-FITC monoclonar antibodies and abiotinylated anti-lipocalin antiserum followed by streptavidin-PE.Results for the wild-type lipocalin and the IL-4 receptor alpha bindinglipocalin mutein S191.4 B24 are shown in FIGS. 39A and 39B,respectively. The determined percentage of PE-positive B cells wasfitted against the concentration of the lipocalin muteins (FIG. 39C) andthe EC₅₀ calculated from the obtained curve. The EC₅₀ of the IL-4receptor alpha binding mutein S191.4 B24 (SEQ ID NO:4) for binding toprimary B cells was calculated as 105 pM.

Example 51 Bioavailability of the Muteins after Subcutaneous andIntratracheal Administration

The bioavailability of the the IL-4 receptor alpha binding mutein S191.4B24 was determined after intravenous, subcutaneous or intratrachealadministration, by monitoring the plasma concentrations of the muteinS191.4 B24 for 4 hours after a 4 mg/kg bolus injection in rats.Intratracheal administration was carried out using a commerciallyavailable intratrachial dosing device (MicroSprayer®, Penn-Century Inc,Philiadelphia, Pa., USA) that generates an aerosol from the tip of along, thin tube attached to a syringe. The aerosol size was about 20 μm.The results of the non-compartmental pharmacokinetic (PK) analysisdemonstrate 100% bioavailability upon subcutaneous injection and that,in contrast to antibodies, the pulmonary delivery of the human tearlipocalin muteins appears to be feasible. The obtained results are shownin Table XV.

TABLE XV Half-life and bioavailability of S191.4 B24 after intravenous(i.v.), subcutaneous (s.c.) and intratracheal (i.t.) administration.i.v. s.c. i.t. t_(1/2) [h] 0.78 1.6 2.36 bioavailability (AUC_(last))n/a 97.2%   10% bioavailability (AUC_(inf)) n/a  119% 13.8%

Example 52 In Vitro Potency of PEGylated VEGF Antagonists Using a HUVECProliferation Assay

Inhibition of VEGF stimulated HUVEC cell proliferation was assessedessentially as described in Example 20 with the following modifications:The VEGF-specific mutein 5236.1-A22 (SEQ ID NO:44) was coupled to PEG20, PEG 30 or PEG 40 at position 95C as described in Example 28 above.The mutein, its PEGylated derivatives and wildtype tear lipocalin (geneproduct of pTLPC26; as control) were added in a dilution series toVEGF165 and incubated for 30 min. at room temperature. The mixtures wereadded to HUVEC cells in triplicate wells to yield a final concentrationof 20 ng/ml VEGF and concentrations between 0.003 nM and 2,000 nM asindicated. Viability of the cells was assessed after 6 days withCellTiter-Glo (Promega) according to the manufacturer's instructions.

Results from measurements employing the above-mentioned muteins areshown in FIG. 41. S236.1-A22 (SEQ ID NO:44) and its PEGylatedderivatives show marked inhibition of VEGF-induced proliferation ofHUVEC cells decreasing with the molecular weight of the attached PEGmoiety, whereas wildtype tear lipocalin does not inhibit VEGF-inducedcell proliferation (Table XVI).

TABLE XVI IC₅₀ values of S236.1-A22 (SEQ ID NO: 44) and its derivativesPEGylated with PEG 20, PEG 30 or PEG 40 for HUVEC cell proliferationinhibition. IC₅₀ (nM) S236.1-A22 0.4 S236.1-A22-PEG20 0.53S236.1-A22-PEG30 2.13 S236.1-A22-PEG40 3.27

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention. Theinvention has been described broadly and generically herein. Each of thenarrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Further embodiments of the invention willbecome apparent from the following claims.

We claim:
 1. A method of binding a non-natural ligand of human tearlipocalin in a subject in need thereof, comprising administering to thesubject a mutein of human tear lipocalin or a pharmaceutical compositioncomprising such mutein, wherein at least one of the cysteine residuesoccurring at sequence positions 61 and 153 of the linear wild type aminoacid sequence of mature human tear lipocalin (SEQ ID NO: 71) is replacedby other amino acids and wherein at least 12 mutated amino acid residuesare present at any of the sequence positions 26-34, 56-58, 80, 83,104-106, and 108 of the linear wild type amino acid sequence of maturehuman tear lipocalin.
 2. The method of claim 1, wherein the non-naturalligand is selected from the group consisting of a peptide, a protein, afragment or a domain of a protein, and a small organic molecule orwherein said peptide includes an amyloid beta peptide and said proteinincludes fibronectin or a domain thereof.
 3. The method of claim 1,wherein the non-natural ligand is selected from the group consisting ofinterleukin 4 receptor alpha chain (IL-4 receptor alpha), vascularendothelial growth factor receptor 2 (VEGF-R2) and vascular endothelialgrowth factor (VEGF).
 4. The method of claim 1, wherein the mutein isconjugated to or at its N-terminus or its C-terminus operably fused to amember selected from the group consisting of an enzyme, a toxin, aprotein or a protein domain, a peptide, a signal sequence and anaffinity tag.
 5. The method of claim 1, wherein the mutein is employedin conjugated form or as a fusion protein.
 6. The method of claim 1,wherein the mutein is conjugated to or at its N-terminus or itsC-terminus operably fused to an antibody or a mutein of human lipocalin.7. The method of claim 1, wherein the mutein is fused to a moiety thatextends the serum half-life of the mutein.
 8. The method of claim 7,wherein the moiety that extends the serum half-life is selected from thegroup consisting of an Fc part of an immunoglobulin, a CH3 domain of animmunoglobulin, a CH4 domain of an immunoglobulin, albumin or an albuminfragment, an albumin binding peptide, an albumin binding protein andtransferrin.
 9. The method of claim 1, wherein the mutein is conjugatedto a label selected from the groups consisting of organic molecules,enzyme labels, radioactive labels, fluorescent labels, chromogeniclabels, luminescent labels, haptens, digoxigenin, biotin, metalcomplexes, metals, colloidal gold and a moiety that extends the serumhalf-life of the mutein.
 10. The method of claim 1, wherein the muteinhas an amino acid sequence as set forth in any one of SEQ ID NOs: 2-8,26-33 and 44-47.